Ligand screening and discovery

ABSTRACT

Disclosed is a method that includes: (i) providing a plurality of initial nucleic acid cassettes that include: a) a first coding region encoding a first immunoglobulin variable domain, b) a second coding region encoding a second immunoglobulin variable domain, and c) a ribosomal binding site disposed between the first and second coding regions for translation of the second polypeptide in a first expression system, wherein the first and second coding regions are in the same translational orientation; (ii) modifying each nucleic acid cassette of the plurality in a single reaction mixture so that it is functional in a second expression system, wherein the first and second region remain physically attached during the modifying; (iii) introducing each modified nucleic acid cassette into a mammalian cell to produce a mixture of transfected cells; and (iv) expressing each modified nucleic acid cassette in the transfected cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. ProvisionalPatent Application Serial No. 60/362,403, filed Mar. 7, 2002, thecontents of which are incorporated by reference in its entirety.

BACKGROUND

[0002] Recombinant techniques have allowed the discovery of artificialand natural proteins that have broad applications in the development oftherapeutics, diagnostic agents (e.g., for imaging or binding assays),enzymes, and agents for affinity separations. One such recombinanttechnique is the construction of nucleic acid libraries that includediverse sequence content. Libraries can be screened by hybridization,genetic complementation, and polypeptide expression, among otheractivities. One challenge for the development of recombinant proteins isthe rapid identification of proteins that are functional in the contextof their ultimate therapeutic or industrial use.

[0003] One exemplary class of proteins being developed as therapeuticsincludes antibodies. The properties of antibodies are being exploited inorder to design agents that bind to human target molecules, so-called,“self-antigens.” For example, a number of monospecific antibodies havebeen approved as human therapeutics. These include Orthoclone OKT3,which targets CD3 antigen; ReoPro, which targets GP IIb/IIIa; Rituxan,which targets CD20; Zenapax and Simulect, which target interleukin-2receptors; Herceptin, which targets the HER2-receptor; Remicade, whichtargets tumor necrosis factor; Synagis, which targets the F protein ofrespiratory syncytial virus; Mylotarg, which targets CD33; and Campath,which targets CD52 (see, e.g., Carter (2001) Nature Reviews 1:118-129;Ezzell (2001) Scientific American October 2001, pages 36-41; Garber(2001) Nat. Biotechnol. 19:184-185).

SUMMARY

[0004] Nucleic acids encoding hetero-oligomeric receptors arereformatted to facilitate shuttling between expression systems. Themethods can be used, for example, to effectively transition multiplecandidates from a first library screen to a biological screen or otherregime.

[0005] In one aspect, the invention features a method that includes: (i)providing a plurality of initial nucleic acid cassettes that include a)a first coding region encoding a first immunoglobulin variable domain,b) a second coding region encoding a second immunoglobulin variabledomain, and c) a ribosomal binding site disposed between the first andsecond coding regions for translation of the second polypeptide in afirst expression system, wherein the first and second coding regions arein the same translational orientation, and the first and second codingregions encode at least segments of subunits of an antigen bindingprotein; (ii) modifying each nucleic acid cassette of the plurality in asingle reaction mixture so that it is functional in a second expressionsystem, wherein the first and second region remain physically attachedduring the modifying; (iii) introducing each modified nucleic acidcassette into a mammalian cell to produce a mixture of transfectedcells; and (iv) screening the mixture of transfected cells using FACS toidentify transfected cell that produces a least a threshold amount of afull Ig that includes the combination of first and second immunoglobulinvariable domain present in an initial cassette. The method can be usedfor expressing a hetero-multimeric protein, e.g., an antigen bindingprotein such as a Fab and/or a full length antibody, a T cell receptor,an MHC complex, and so forth.

[0006] The first and second coding regions can be transcribed as asingle transcript. In one embodiment, prior to the modifying, thenucleic acid cassette includes a promoter (e.g., a prokaryotic promoter)that regulates transcription of a single transcript that includes thefirst and second coding regions and is disposed upstream of the firstcoding region. The regions can further include a sequence encoding animmunoglobulin constant domain (e.g., CL or CH1).

[0007] In one embodiment, the first expression system is prokaryotic andthe second expression system is eukaryotic. For example, the secondexpression system is mammalian or fungal (e.g., yeast). In a particularexample, the first expression system is prokaryotic (e.g., filamentousbacteriophage display) and the second expression system is mammalian. Inanother particular example, the first expression system is prokaryotic(e.g., filamentous bacteriophage display) and the second expressionsystem is yeast.

[0008] In another embodiment, the first and second expression systemsare eukaryotic. For example, the first expression system is yeast andthe second expression system is mammalian.

[0009] In another embodiment, one of the expression systems can also bean in vitro translation system, e.g., an in vitrotranscription-translation system or an eukaryotic in vitro translationsystem.

[0010] The modifying can include inserting an internal ribosome entrysite between the first and second coding regions. The modifying can alsoinclude removing a segment that includes the ribosomal binding site.

[0011] The modifying can include linking a eukaryotic promoter to thecassette upstream of the first coding region such that the promoterregulates transcription of a transcript that includes the first codingregion or the first and second coding region. In one example, themodifying can further include removing the prokaryotic promoter (beforeor after the inserting). In another example, the prokaryotic promoter isretained, and, e.g., the eukaryotic promoter is linked upstream,downstream, or within the eukaryotic promoter.

[0012] The eukaryotic promoter may be functional, for example, in amammalian, yeast, and/or plant cell, e.g., a human cell or aSaccharomyces cell.

[0013] The modifying can include linking (e.g., inserting) a segmentthat includes a eukaryotic promoter between the first and second codingregion. The eukaryotic promoter linked at this position is typically inaddition to the eukaryotic promoter positioned upstream of the cassette.The segment can also include a leader, e.g., a leader that supportseukaryotic processing, e.g., a bifunctional leader or an exclusivelyeukaryotic leader.

[0014] The modifying can include linking (e.g., inserting) a segmentthat includes an internal ribosome entry site (IRES) (e.g., a viralIRES) between the first and second coding regions.

[0015] The segment can further include a signal sequence functional in amammalian cell. The segment can further include a polyA additionregulatory sequence. In one embodiment, the segment includes both aeukaryotic polyA addition regulatory sequence and a eukaryotic promoter,e.g., a second eukaryotic promoter.

[0016] In a related embodiment, the segment includes a signal sequencefunctional in a mammalian cell.

[0017] In one embodiment, prior to or after the modifying, the secondcoding region of the nucleic acid cassette is in frame with a sequenceencoding a first fusion polypeptide. For example, the first fusionpolypeptide can include the polypeptide encoded by the first codingregion and a bacteriophage coat protein or fragment thereof (e.g., aminor coat protein such as gene III or the gene III stump.).

[0018] In one example, the modifying includes removing the sequenceencoding the first fusion polypeptide, e.g., removing a sequence thatencodes a bacteriophage coat protein or fragment thereof. The modifyingcan include attaching a different sequence encoding a second fusionpolypeptide in frame to the second coding region. For example, thesecond fusion polypeptide may include an immunoglobulin heavy chain CH2and CH3, Hinge-CH2-CH3, or other constant domains. The Fe region, whichtypically includes Hinge-CH2-CH3 can also include a modification thatalters binding to an Fe receptor relative to another Fe receptor. Thesecond polypeptide can further include other functional domains, e.g., anon-immunoglobulin functional domain, e.g., a localization signal, e.g.,a cell attachment sequence. The second polypeptide can alsoindependently include a polypeptide tag. In one embodiment, the first orsecond fusion includes a yeast extracellular domain or portion thereof.

[0019] In an embodiment, as a (direct or indirect) fusion of one of theimmunoglobulin variable domains to the Fe domain includes a sequencemodification that alters binding to an Fe receptor relative to anotherFe receptor, e.g., the Fe domain is artificial and selectively binds toFe receptors.

[0020] In one embodiment, the modifying includes (in any order, orconcurrently) a) replacing a nucleic acid segment between the first andsecond coding regions, b) replacing nucleic acid 5′ of the coding strandof the first coding region and c) replacing nucleic acid 3′ of thecoding strand of the second coding region. For example, b) may be c)concurrent.

[0021] In one embodiment, the nucleic acid 5′ of the coding strand isdirectly adjacent to the first coding region.

[0022] The modifying can include adding or removing one or morenucleotides but maintaining linkage between the first and second codingregions. With respect to the coding strand, the modifying can include:a) maintaining linkage between the 5′ terminus of the first codingregion and the 3′ terminus of the second coding region, while disruptingthe linkage between the 3′ terminus of the first coding region and the5′ terminus of the second coding region; and b) maintaining linkagebetween the 5′ terminus of the first coding region and the 3′ terminusof the second coding region, while disrupting the linkage between the 3′terminus of the first coding region and the 5′ terminus of the secondcoding region. The order can be reversed.

[0023] The nucleic acid 5′ of the coding strand can include one or moreof: an untranslated region, a ribosome binding site, a promoter, asegment encoding a leader sequence or portion thereof.

[0024] In another embodiment, the modifying includes only a singlecloning step. The segment between the first and second coding regionsincludes a sequence encoding a signal sequence that is functional in twodifferent expression systems. The signal sequence is fused to the secondcoding region. For example, the signal sequence is functional in bothprokaryotic and eukaryotic cells, e.g., it includes VHA at the −3, −2,−1 positions, or it has at least 80%, 90%, 95%, or 100% identity to anatural signal sequence that is functional in both systems, e.g., abeta-lactamase signal sequence. A single cloning step can be used toalter the in-frame fusion of the second coding region and a third codingregion (e.g., encoding an Fe domain or a gene III protein or fragmentthereof).

[0025] The modifying can include annealing and extending a primer thatincludes a restriction site, e.g., that is endogenous or exogenous tothe nucleic acid cassette prior to the modifying. For example, PCR canbe used.

[0026] The modifying can include restricting the cassette using one ormore of: ApaLI, AscI, MfeI, BstEII, NotI, XbaI, NcoI, PstI, NheI, SfiIand BssH2, e.g., including combinations such as AscI and MfeI; AscI andSfiI; ApaL1 and NotI; ApaL1 and NheI; or ApaL1 and BstEII.

[0027] In one embodiment, the VL domain of the expressed heteromericprotein includes a naturally occurring N-terminus after leader sequenceprocessing.

[0028] The modified nucleic acid cassette can include a chromatincontrol sequence (e.g., an insulator, a locus control region, or achromatin opening element). The chromatin control sequence can beupstream of the first coding region, e.g., upstream of a promoter thatis operably linked to the first coding region, or down stream of thesecond coding region, etc. The control sequence might even be insertedbetween the first and second coding regions.

[0029] In another aspect, the invention features a method that includes:(i) providing a plurality of nucleic acid cassettes, each nucleic acidcassette that includes a) a first coding region encoding a firstpolypeptide, b) a second coding region encoding a second polypeptide,and c) a ribosomal binding site disposed between the first and secondcoding regions for translation of the second polypeptide in a firstexpression system, wherein the first and second coding regions are inthe same translational orientation, and the first and second codingregions can be transcribed as a single transcript; and the first andsecond coding regions encode at least segments of subunits of ahetero-multimeric protein, and (ii) modifying each nucleic acid cassetteof the plurality to produce second nucleic acids, each second nucleicacid being functional in a second expression system, wherein the firstand second coding regions remain attached. The method can be used toreformat selected nucleic acids in bulk (e.g., en masse or individuallyin parallel) or to reformat a library. For example, the plurality caninclude between 10² and 10⁸ members, e.g., 10²-10⁵, 10³-10⁵, or 5-100.Each linkage of a first and second coding regions from the first nucleicacids can be represented among the second nucleic acids.

[0030] The modifying can include releasing, from first vectors, nucleicacid fragment that include the first and second coding regions andinserting the nucleic acid fragments into second vectors. The providingof first nucleic acids can include selecting members of a displaylibrary (e.g., a phage or yeast display library) for binding to atarget.

[0031] The method can be used for expressing hetero-multimeric proteins,e.g., an antigen binding protein such as a Fab and/or a full lengthantibody, a T cell receptor, an MHC complex, and so forth.

[0032] Each of the first and second coding regions can be transcribed asa single transcript. In one embodiment, prior to the modifying, thenucleic acid cassettes include a promoter (e.g., a prokaryotic promoter)that regulates transcription of a single transcript that includes thefirst and second coding regions and is disposed upstream of the firstcoding region.

[0033] In one embodiment, the first expression system is prokaryotic andthe second expression system is eukaryotic. For example, the secondexpression system is mammalian or fungal (e.g., yeast). In a particularexample, the first expression system is prokaryotic (e.g., filamentousbacteriophage display) and the second expression system is mammalian. Inanother particular example, the first expression system is prokaryotic(e.g., filamentous bacteriophage display) and the second expressionsystem is yeast.

[0034] In another embodiment, the first and second expression systemsare eukaryotic. For example, the first expression system is yeast andthe second expression system is mammalian.

[0035] In another embodiment, one of the expression systems can also bean in vitro translation system, e.g., an in vitrotranscription-translation system or an eukaryotic in vitro translationsystem.

[0036] For each nucleic acid cassette of the plurality, the modifyingcan include inserting an internal ribosome entry site between the firstand second coding regions. The modifying can also include removing asegment that includes the ribosomal binding site.

[0037] For each nucleic acid cassette of the plurality, the modifyingcan include linking a eukaryotic promoter to the cassette upstream ofthe first coding region such that the promoter regulates transcriptionof a transcript that includes the first coding region or the first andsecond coding region. In one example, for each nucleic acid cassette ofthe plurality, the modifying can further include removing theprokaryotic promoter (before or after the inserting). In anotherexample, the prokaryotic promoter is retained, and, e.g., the eukaryoticpromoter is linked upstream, downstream, or within the eukaryoticpromoter.

[0038] The eukaryotic promoter may be functional, for example, in amammalian, yeast, and/or plant cell, e.g., a human cell or aSaccharomyces cell.

[0039] For each nucleic acid cassette of the plurality, the modifyingcan include linking (e.g., inserting) a segment that includes aeukaryotic promoter between the first and second coding region. Theeukaryotic promoter linked at this position is typically in addition tothe eukaryotic promoter positioned upstream of the cassette. The segmentcan also include a leader, e.g., a leader that supports eukaryoticprocessing, e.g., a bifunctional leader or an exclusively eukaryoticleader.

[0040] For each nucleic acid cassette of the plurality, the modifyingcan include linking (e.g., inserting) a segment that includes aninternal ribosome entry site (IRES) (e.g., a viral IRES) between thefirst and second coding regions.

[0041] Each of the segments can further include a signal sequencefunctional in a mammalian cell. The segment can further include a polyAaddition regulatory sequence. In one embodiment, the segment includesboth a eukaryotic polyA addition regulatory sequence and a eukaryoticpromoter, e.g., a second eukaryotic promoter.

[0042] In a related embodiment, the segment includes a signal sequencefunctional in a mammalian cell.

[0043] In one embodiment, prior to or after the modifying, the secondcoding region of the nucleic acid cassette is in frame with a sequenceencoding a first fusion polypeptide. For example, the first fusionpolypeptide can include the polypeptide encoded by the first codingregion and a bacteriophage coat protein or fragment thereof (e.g., aminor coat protein such as gene III or the gene III stump.).

[0044] In one example, for each nucleic acid cassette of the plurality,the modifying includes removing the sequence encoding the first fusionpolypeptide, e.g., removing a sequence that encodes a bacteriophage coatprotein or fragment thereof. The modifying can include attaching adifferent sequence encoding a second fusion polypeptide in frame to thesecond coding region. For example, the second fusion polypeptide mayinclude an immunoglobulin heavy chain CH2 and CH3, Hinge-CH2-CH3, orother constant domains. The Fc region, which typically includesHinge-CH2-CH3, can also include a modification that alters binding to anFc receptor relative to another Fc receptor. The second polypeptide canfurther include other functional domains, e.g., a non-immunoglobulinfunctional domain, e.g., a localization signal, e.g., a cell attachmentsequence. The second polypeptide can also independently include apolypeptide tag. In one embodiment, the first or second fusion includesa yeast extracellular domain or portion thereof.

[0045] The regions can further include a sequence encoding animmunoglobulin constant domain (e.g., CL or CH1).

[0046] In an embodiment, as a (direct or indirect) fusion of one of theimmunoglobulin variable domains to the Fc domain includes a sequencemodification that alters binding to an Fc receptor relative to anotherFc receptor, e.g., the Fc domain is artificial and selectively binds toFc receptors.

[0047] In one embodiment, for each nucleic acid cassette of theplurality, the modifying includes (in any order, or concurrently) a)replacing a nucleic acid segment between the first and second codingregions, b) replacing nucleic acid 5′ of the coding strand of the firstcoding region and c) replacing nucleic acid 3′ of the coding strand ofthe second coding region. For example, b) may be c) concurrent.

[0048] In one embodiment, the nucleic acid 5′ of the coding strand isdirectly adjacent to the first coding region.

[0049] The modifying can include adding or removing one or morenucleotides but maintaining linkage between the first and second codingregions. With respect to the coding strand, the modifying can include:a) maintaining linkage between the 5′ terminus of the first codingregion and the 3′ terminus of the second coding region, while disruptingthe linkage between the 3′ terminus of the first coding region and the5′ terminus of the second coding region; and b) maintaining linkagebetween the 5′ terminus of the first coding region and the 3′ terminusof the second coding region, while disrupting the linkage between the 3′terminus of the first coding region and the 5′ terminus of the secondcoding region. The order can be reversed.

[0050] The nucleic acid 5′ of the coding strand can include one or moreof: an untranslated region, a ribosome binding site, a promoter, asegment encoding a leader sequence or portion thereof.

[0051] In another embodiment, the modifying includes only a singlecloning step. The segment between the first and second coding regionsincludes a sequence encoding a signal sequence that is functional in twodifferent expression systems. The signal sequence is fused to the secondcoding region. For example, the signal sequence is functional in bothprokaryotic and eukaryotic cells, e.g., it includes VHA at the −3, −2,−1 positions, or it has at least 80%, 90%, 95%, or 100% identity to anatural signal sequence that is functional in both systems, e.g., abeta-lactamase signal sequence. A single cloning step can be used toalter the in-frame fusion of the second coding region and a third codingregion (e.g., encoding an Fe domain or a gene III protein or fragmentthereof).

[0052] The modifying can include annealing and extending a primer thatincludes a restriction site, e.g., that is endogenous or exogenous tothe nucleic acid cassette prior to the modifying. For example, PCR canbe used.

[0053] The modifying can include restricting the cassette using one ormore of: ApaLI, AscI, MfeI, BstEII, NotI, XbaI, NeoI, PstI, NheI, SfiIand BssH2, e.g., including combinations such as AscI and MfeI; AscI andSfiI;ApaL1 and NotI; ApaL1 and NheI; or ApaL1 and BstEII.

[0054] The first and/or second polypeptide domain can include a leadersequence junction which is functional (e.g., cleavable) by bothprokaryotic and eukaryotic cells. (The junction being only a region ofthe leader sequence that is directly N-terminal to the cleavage site,e.g., the 5, 4, or 3 amino acids N-terminal to the cleavage site.). Forexample, the leader sequence junction, e.g., includes at positions −3,−2, and −1: Val-His-Ala.

[0055] In one embodiment, the VL domain of the expressed heteromericprotein includes a naturally occurring N-terminus after leader sequenceprocessing.

[0056] Each of the modified nucleic acid cassettes can include achromatin control sequence (e.g., an insulator, a locus control region,or a chromatin opening element). The chromatin control sequence can beupstream of the first coding region, e.g., upstream of a promoter thatis operably linked to the first coding region, or down stream of thesecond coding region, etc. The control sequence might even be insertedbetween the first and second coding regions.

[0057] In one aspect, the invention features a method that includes: (i)providing a nucleic acid cassette that includes a) a first coding regionencoding a first polypeptide, b) a second coding region encoding asecond polypeptide, and c) a ribosomal binding site disposed between thefirst and second coding regions for translation of the secondpolypeptide in a first expression system, wherein the first and secondcoding regions are in the same translational orientation, and the firstand second coding regions encode at least segments of subunits of thehetero-multimeric protein; (ii) modifying the nucleic acid cassette sothat it is functional in a second expression system, wherein the firstand second region remain physically attached during the modifying; and(iii) expressing the hetero-multimeric protein from the modified nucleicacid cassette in the second expression system. The method can be usedfor expressing a hetero-multimeric protein, e.g., an antigen bindingprotein such as a Fab and/or a full length antibody, a T cell receptor,an MHC complex, and so forth.

[0058] The first and second coding regions can be transcribed as asingle transcript. In one embodiment, prior to the modifying, thenucleic acid cassette includes a promoter (e.g., a prokaryotic promoter)that regulates transcription of a single transcript that includes thefirst and second coding regions and is disposed upstream of the firstcoding region.

[0059] In one embodiment, the first expression system is prokaryotic andthe second expression system is eukaryotic. For example, the secondexpression system is mammalian or fungal (e.g., yeast). In a particularexample, the first expression system is prokaryotic (e.g., filamentousbacteriophage display) and the second expression system is mammalian. Inanother particular example, the first expression system is prokaryotic(e.g., filamentous bacteriophage display) and the second expressionsystem is yeast.

[0060] In another embodiment, the first and second expression systemsare eukaryotic. For example, the first expression system is yeast andthe second expression system is mammalian.

[0061] In another embodiment, one of the expression systems can also bean in vitro translation system, e.g., an in vitrotranscription-translation system or an eukaryotic in vitro translationsystem.

[0062] The modifying can include inserting an internal ribosome entrysite between the first and second coding regions. The modifying can alsoinclude removing a segment that includes the ribosomal binding site.

[0063] The modifying can include linking a eukaryotic promoter to thecassette upstream of the first coding region such that the promoterregulates transcription of a transcript that includes the first codingregion or the first and second coding region. In one example, themodifying can further include removing the prokaryotic promoter (beforeor after the inserting). In another example, the prokaryotic promoter isretained, and, e.g., the eukaryotic promoter is linked upstream,downstream, or within the eukaryotic promoter.

[0064] The eukaryotic promoter may be functional, for example, in amammalian, yeast, and/or plant cell, e.g., a human cell or aSaccharomyces cell.

[0065] The modifying can include linking (e.g., inserting) a segmentthat includes a eukaryotic promoter between the first and second codingregion. The eukaryotic promoter linked at this position is typically inaddition to the eukaryotic promoter positioned upstream of the cassette.The segment can also include a leader, e.g., a leader that supportseukaryotic processing, e.g., a bifunctional leader or an exclusivelyeukaryotic leader.

[0066] The modifying can include linking (e.g., inserting) a segmentthat includes an internal ribosome entry site (IRES) (e.g., a viralIRES) between the first and second coding regions.

[0067] The segment can further include a signal sequence functional in amammalian cell. The segment can further include a polyA additionregulatory sequence. In one embodiment, the segment includes both aeukaryotic polyA addition regulatory sequence and a eukaryotic promoter,e.g., a second eukaryotic promoter.

[0068] In a related embodiment, the segment includes a signal sequencefunctional in a mammalian cell.

[0069] In one embodiment, prior to or after the modifying, the secondcoding region of the nucleic acid cassette is in frame with a sequenceencoding a first fusion polypeptide. For example, the first fusionpolypeptide can include the polypeptide encoded by the first codingregion and a bacteriophage coat protein or fragment thereof (e.g., aminor coat protein such as gene III or the gene III stump.).

[0070] In one example, the modifying includes removing the sequenceencoding the first fusion polypeptide, e.g., removing a sequence thatencodes a bacteriophage coat protein or fragment thereof. The modifyingcan include attaching a different sequence encoding a second fusionpolypeptide in frame to the second coding region. For example, thesecond fusion polypeptide may include an immunoglobulin heavy chain CH2and CH3, Hinge-CH2-CH3, or other constant domains. The Fc region, whichtypically includes Hinge-CH2-CH3 can also include a modification thatalters binding to an Fc receptor relative to another Fc receptor. Thesecond polypeptide can further include other functional domains, e.g., anon-immunoglobulin functional domain, e.g., a localization signal, e.g.,a cell attachment sequence. The second polypeptide can alsoindependently include a polypeptide tag. In one embodiment, the first orsecond fusion includes a yeast extracellular domain or portion thereof.

[0071] In one embodiment, the first and second coding regions encodeimmunoglobulin variable domain, e.g., respectively, a VH and VL or VLand VH domains. The regions can further include a sequence encoding animmunoglobulin constant domain (e.g., CL or CH1).

[0072] In an embodiment, as a (direct or indirect) fusion of one of theimmunoglobulin variable domains to the Fc domain includes a sequencemodification that alters binding to an Fc receptor relative to anotherFc receptor, e.g., the Fe domain is artificial and selectively binds toFe receptors.

[0073] In one embodiment, the modifying includes (in any order, orconcurrently) a) replacing a nucleic acid segment between the first andsecond coding regions, b) replacing nucleic acid 5′ of the coding strandof the first coding region and c) replacing nucleic acid 3′ of thecoding strand of the second coding region. For example, b) may be c)concurrent.

[0074] In one embodiment, the nucleic acid 5′ of the coding strand isdirectly adjacent to the first coding region.

[0075] The modifying can include adding or removing one or morenucleotides but maintaining linkage between the first and second codingregions. With respect to the coding strand, the modifying can include:a) maintaining linkage between the 5′ terminus of the first codingregion and the 3′ terminus of the second coding region, while disruptingthe linkage between the 3′ terminus of the first coding region and the5′ terminus of the second coding region; and b) maintaining linkagebetween the 5′ terminus of the first coding region and the 3′ terminusof the second coding region, while disrupting the linkage between the 3′terminus of the first coding region and the 5′ terminus of the secondcoding region. The order can be reversed.

[0076] The nucleic acid 5′ of the coding strand can include one or moreof: an untranslated region, a ribosome binding site, a promoter, asegment encoding a leader sequence or portion thereof.

[0077] In another embodiment, the modifying includes only a singlecloning step. The segment between the first and second coding regionsincludes a sequence encoding a signal sequence that is functional in twodifferent expression systems. The signal sequence is fused to the secondcoding region. For example, the signal sequence is functional in bothprokaryotic and eukaryotic cells, e.g., it includes VHA at the −3, −2,−1 positions, or it has at least 80%, 90%, 95%, or 100% identity to anatural signal sequence that is functional in both systems, e.g., abeta-lactamase signal sequence. A single cloning step can be used toalter the in-frame fusion of the second coding region and a third codingregion (e.g., encoding an Fc domain or a gene III protein or fragmentthereof).

[0078] The modifying can include annealing and extending a primer thatincludes a restriction site, e.g., that is endogenous or exogenous tothe nucleic acid cassette prior to the modifying. For example, PCR canbe used.

[0079] The modifying can include restricting the cassette using one ormore of: ApaLI, AscI, MfeI, BstEII, NotI, XbaI, NcoI, PstI, NheI, SfiIand BssH2, e.g., including combinations such as AscI and MfeI; AscI andSfiI;ApaL1 and NotI; ApaL1 and NheI; or ApaL1 and BstEII.

[0080] The first and/or second polypeptide domain can include a leadersequence junction which is functional (e.g., cleavable) by bothprokaryotic and eukaryotic cells. (The junction being only a region ofthe leader sequence that is directly N-terminal to the cleavage site,e.g., the 5, 4, or 3 amino acids N-terminal to the cleavage site.). Forexample, the leader sequence junction, e.g., includes at positions −3,−2, and −1: Val-His-Ala.

[0081] In one embodiment, the VL domain of the expressed heteromericprotein includes a naturally occurring N-terminus after leader sequenceprocessing.

[0082] The modified nucleic acid cassette can include a chromatincontrol sequence (e.g., an insulator, a locus control region, or achromatin opening element). The chromatin control sequence can beupstream of the first coding region, e.g., upstream of a promoter thatis operably linked to the first coding region, or down stream of thesecond coding region, etc. The control sequence might even be insertedbetween the first and second coding regions.

[0083] In another aspect, the invention features a method that includes:(i) providing a plurality of nucleic acid cassettes, each nucleic acidcassette that includes a) a first coding region encoding a firstpolypeptide, b) a second coding region encoding a second polypeptide,and c) a ribosomal binding site disposed between the first and secondcoding regions for translation of the second polypeptide in a firstexpression system, wherein the first and second coding regions are inthe same translational orientation, and the first and second codingregions can be transcribed as a single transcript; and the first andsecond coding regions encode at least segments of subunits of ahetero-multimeric protein, and (ii) modifying each nucleic acid cassetteof the plurality to produce second nucleic acids, each second nucleicacid being functional in a second expression system, wherein the firstand second coding regions remain attached. The method can be used toreformat selected nucleic acids in bulk (e.g., en masse or individuallyin parallel) or to reformat a library. For example, the plurality caninclude between 10² and 10⁸ members, e.g., 10²-10⁵, 10³-10⁵, or 5-100.Each linkage of a first and second coding regions from the first nucleicacids can be represented among the second nucleic acids.

[0084] The modifying can include releasing, from first vectors, nucleicacid fragment that include the first and second coding regions andinserting the nucleic acid fragments into second vectors. The providingof first nucleic acids can include selecting members of a displaylibrary (e.g., a phage or yeast display library) for binding to atarget.

[0085] The method can be used for expressing hetero-multimeric proteins,e.g., an antigen binding protein such as a Fab and/or a full lengthantibody, a T cell receptor, an MHC complex, and so forth.

[0086] Each of the first and second coding regions can be transcribed asa single transcript. In one embodiment, prior to the modifying, thenucleic acid cassettes include a promoter (e.g., a prokaryotic promoter)that regulates transcription of a single transcript that includes thefirst and second coding regions and is disposed upstream of the firstcoding region.

[0087] In one embodiment, the first expression system is prokaryotic andthe second expression system is eukaryotic. For example, the secondexpression system is mammalian or fungal (e.g., yeast). In a particularexample, the first expression system is prokaryotic (e.g., filamentousbacteriophage display) and the second expression system is mammalian. Inanother particular example, the first expression system is prokaryotic(e.g., filamentous bacteriophage display) and the second expressionsystem is yeast.

[0088] In another embodiment, the first and second expression systemsare eukaryotic. For example, the first expression system is yeast andthe second expression system is mammalian.

[0089] In another embodiment, one of the expression systems can also bean in vitro translation system, e.g., an in vitrotranscription-translation system or an eukaryotic in vitro translationsystem.

[0090] For each nucleic acid cassette of the plurality, the modifyingcan include inserting an internal ribosome entry site between the firstand second coding regions. The modifying can also include removing asegment that includes the ribosomal binding site.

[0091] For each nucleic acid cassette of the plurality, the modifyingcan include linking a eukaryotic promoter to the cassette upstream ofthe first coding region such that the promoter regulates transcriptionof a transcript that includes the first coding region or the first andsecond coding region. In one example, for each nucleic acid cassette ofthe plurality, the modifying can further include removing theprokaryotic promoter (before or after the inserting). In anotherexample, the prokaryotic promoter is retained, and, e.g., the eukaryoticpromoter is linked upstream, downstream, or within the eukaryoticpromoter.

[0092] The eukaryotic promoter may be functional, for example, in amammalian, yeast, and/or plant cell, e.g., a human cell or aSaccharomyces cell.

[0093] For each nucleic acid cassette of the plurality, the modifyingcan include linking (e.g., inserting) a segment that includes aeukaryotic promoter between the first and second coding region. Theeukaryotic promoter linked at this position is typically in addition tothe eukaryotic promoter positioned upstream of the cassette. The segmentcan also include a leader, e.g., a leader that supports eukaryoticprocessing, e.g., a bifunctional leader or an exclusively eukaryoticleader.

[0094] For each nucleic acid cassette of the plurality, the modifyingcan include linking (e.g., inserting) a segment that includes aninternal ribosome entry site (IRES) (e.g., a viral IRES) between thefirst and second coding regions.

[0095] Each of the segments can further include a signal sequencefunctional in a mammalian cell. The segment can further include a polyAaddition regulatory sequence. In one embodiment, the segment includesboth a eukaryotic polyA addition regulatory sequence and a eukaryoticpromoter, e.g., a second eukaryotic promoter.

[0096] In a related embodiment, the segment includes a signal sequencefunctional in a mammalian cell.

[0097] In one embodiment, prior to or after the modifying, the secondcoding region of the nucleic acid cassette is in frame with a sequenceencoding a first fusion polypeptide. For example, the first fusionpolypeptide can include the polypeptide encoded by the first codingregion and a bacteriophage coat protein or fragment thereof (e.g., aminor coat protein such as gene III or the gene III stump.).

[0098] In one example, for each nucleic acid cassette of the plurality,the modifying includes removing the sequence encoding the first fusionpolypeptide, e.g., removing a sequence that encodes a bacteriophage coatprotein or fragment thereof. The modifying can include attaching adifferent sequence encoding a second fusion polypeptide in frame to thesecond coding region. For example, the second fusion polypeptide mayinclude an immunoglobulin heavy chain CH2 and CH3, Hinge-CH2-CH3, orother constant domains. The Fc region, which typically includesHinge-CH2-CH3 can also include a modification that alters binding to anFc receptor relative to another Fc receptor. The second polypeptide canfurther include other functional domains, e.g., a non-immunoglobulinfunctional domain, e.g., a localization signal, e.g., a cell attachmentsequence. The second polypeptide can also independently include apolypeptide tag. In one embodiment, the first or second fusion includesa yeast extracellular domain or portion thereof.

[0099] In one embodiment, the first and second coding regions encodeimmunoglobulin variable domain, e.g., respectively, a VH and VL or VLand VH domains. The regions can further include a sequence encoding animmunoglobulin constant domain (e.g., CL or CH1).

[0100] In an embodiment, as a (direct or indirect) fusion of one of theimmunoglobulin variable domains to the Fc domain includes a sequencemodification that alters binding to an Fe receptor relative to anotherFe receptor, e.g., the Fe domain is artificial and selectively binds toFe receptors.

[0101] In one embodiment, for each nucleic acid cassette of theplurality, the modifying includes (in any order, or concurrently) a)replacing a nucleic acid segment between the first and second codingregions, b) replacing nucleic acid 5′ of the coding strand of the firstcoding region and c) replacing nucleic acid 3′ of the coding strand ofthe second coding region. For example, b) may be c) concurrent.

[0102] In one embodiment, the nucleic acid 5′ of the coding strand isdirectly adjacent to the first coding region.

[0103] The modifying can include adding or removing one or morenucleotides but maintaining linkage between the first and second codingregions. With respect to the coding strand, the modifying can include:a) maintaining linkage between the 5′ terminus of the first codingregion and the 3′ terminus of the second coding region, while disruptingthe linkage between the 3′ terminus of the first coding region and the5′ terminus of the second coding region; and b) maintaining linkagebetween the 5′ terminus of the first coding region and the 3′ terminusof the second coding region, while disrupting the linkage between the 3′terminus of the first coding region and the 5′ terminus of the secondcoding region. The order can be reversed.

[0104] The nucleic acid 5′ of the coding strand can include one or moreof: an untranslated region, a ribosome binding site, a promoter, asegment encoding a leader sequence or portion thereof.

[0105] In another embodiment, the modifying includes only a singlecloning step. The segment between the first and second coding regionsincludes a sequence encoding a signal sequence that is functional in twodifferent expression systems. The signal sequence is fused to the secondcoding region. For example, the signal sequence is functional in bothprokaryotic and eukaryotic cells, e.g., it includes VHA at the −3, −2,−1 positions, or it has at least 80%, 90%, 95%, or 100% identity to anatural signal sequence that is functional in both systems, e.g., abeta-lactamase signal sequence. A single cloning step can be used toalter the in-frame fusion of the second coding region and a third codingregion (e.g., encoding an Fc domain or a gene III protein or fragmentthereof).

[0106] The modifying can include annealing and extending a primer thatincludes a restriction site, e.g., that is endogenous or exogenous tothe nucleic acid cassette prior to the modifying. For example, PCR canbe used.

[0107] The modifying can include restricting the cassette using one ormore of: ApaLI, AscI, MfeI, BstEII, NotI, XbaI, NcoI, PstI, NheI, SfiIand BssH2, e.g., including combinations such as AscI and MfeI; AscI andSfiI; ApaL1 and NotI; ApaL1 and NheI; or ApaL1 and BstEII.

[0108] The first and/or second polypeptide domain can include a leadersequence junction which is functional (e.g., cleavable) by bothprokaryotic and eukaryotic cells. (The junction being only a region ofthe leader sequence that is directly N-terminal to the cleavage site,e.g., the 5, 4, or 3 amino acids N-terminal to the cleavage site.). Forexample, the leader sequence junction, e.g., includes at positions −3,−2, and −1: Val-His-Ala.

[0109] In one embodiment, the VL domain of the expressed heteromericprotein includes a naturally occurring N-terminus after leader sequenceprocessing.

[0110] Each of the modified nucleic acid cassettes can include achromatin control sequence (e.g., an insulator, a locus control region,or a chromatin opening element). The chromatin control sequence can beupstream of the first coding region, e.g., upstream of a promoter thatis operably linked to the first coding region, or down stream of thesecond coding region, etc. The control sequence might even be insertedbetween the first and second coding regions.

[0111] The invention also features a method of expressing ahetero-multimeric protein. The method includes: (i) providing a firstnucleic acid including a) a first coding region encoding a firstpolypeptide domain, b) a second coding region encoding a secondpolypeptide domain, and c) a sequence encoding a peptide linker thatlinks the first polypeptide domain and the second polypeptide domainwithin a single polypeptide chain, wherein the first and second codingregions are in the same translational orientation, and the first andsecond coding regions encode subunits of an antigen binding protein, and(ii) modifying the first nucleic acid so that the first and secondpolypeptide domains can be translated as separate polypeptides in suchmanner that the DNAs encoding the first and second polypeptide domainsmaintain a physical link throughout the modification procedure. Thefirst and second polypeptide domains can encode an immunoglobulinvariable domain, e.g., VL and VH. The first nucleic acid can encode ascFV. The segment between the first and second coding regions caninclude a peptide linker, e.g., a linker compatible with scFv function.After modification, the sequence can encode, e.g., a Fab or full-chainantibody. The modifying can also be reversed, e.g., to move from amulti-chain format to a single-chain format.

[0112] In still another aspect, the invention features a method thatincludes: providing a first plurality of different nucleic acids, eachencoding a hetero-oligomeric candidate ligand; selecting a subset of thefirst plurality by contacting to a target; reformatting each nucleicacid of the subset for mammalian cell expression, such that each nucleicacid encodes a hetero-oligomeric protein that includes a firstfunctional domain of one subunit of the candidate ligand, a secondfunctional domain of another subunit of the candidate ligand and aneffector domain not encoded by the nucleic acids of the first plurality;introducing members of the subset into a mammalian cell to form aplurality of expression cells that can produce the protein that includesthe functional domain and the effector domain; and screening theexpression cells to identify cells that produce at least a thresholdamount of a ligand-effector domain fusion protein.

[0113] In one embodiment, the introducing is effected separately foreach nucleic acid of the subset or of the first plurality. In anotherembodiment, the introducing includes preparing a mixture that includesnucleic acid for a plurality of members of the subset or of the firstplurality and contacting mammalian cells with the mixture underconditions in which the nucleic acids are introduced into the cells.

[0114] In one embodiment, the screening includes FACS. In anotherembodiment, the screening includes magnetic particle-based separation.The screening can include culturing the cells in a low permeabilitymedium. The screening can further include attaching a probe to surfacesof the cells, the probe having a binding domain that recognizes aconstant region of the fusion proteins, and detecting the amount offusion protein retained by the surface-bound probe. For example, theconstant region can includes a portion of the heavy chain, the lightchain, or combinations thereof. The probe, which includes a protein(e.g., an antibody) that recognizes the constant region of the expressedimmunoglobulin (e.g., an antibody that recognizes human immunoglobulin)may be attached by a linkage that includes a chemical tag, e.g., biotin.The probe can be specific for one of the antibody chains, but not theother (e.g., heavy, but not light; or light but not heavy. The detectingcan includes detecting binding of a label to the other antibody chain(i.e., the one not retained by the surface-bound probe). The detectingcan be specific for whole antibodies.

[0115] In another embodiment, the cells express a heterologous proteinthat is attached to the cell surface and recognizes a constant region ofthe fusion proteins. The heterologous protein can be, e.g. an antibodyspecific for a chain of a human antibody or an Fc receptor, e.g., ahuman Fc receptor or a non-human Fc receptor with specificity for humanFc regions. The effector domain can include CH2 and CH3, e.g., Hinge-CH2and CH3, e.g., an Fc region of a human isotype (e.g., IgG1, etc.isotypes). In one embodiment, each fusion protein of the plurality offusion proteins is glycosylated on Asn 297. In a particular embodiment,each fusion protein can elicit ADCC or CDC.

[0116] Each candidate ligand can includes a plurality of polypeptidechains, e.g., it includes a Fab structure.

[0117] The reformatting is en masse. For example, the reformattingincludes at least a reaction of multiple nucleic acids in the samemixture. The reformatting maintains linkage between the polypeptidechains for each candidate ligand. The introducing and expressing caninclude transient expression and/or generating a stable cell line.

[0118] The expressing can include producing at least 5, 10, 20, 40 mg ofeach fusion protein. At least one of the fusion protein can be expressedin a hollow fiber bioreactor, or a subject organism. In the latter case,the method can include monitoring the subject organism for a clinicalindication, e.g., the subject can be a normal, diseased, ordisease-predisposed individual.

[0119] In an embodiment, a mixture of nucleic acids from the subset areintroduced into the mammalian cells together, e.g., a mixture of nucleicacids is transfected into a population of cells, e.g., under controlledconditions, e.g., under low nucleic acid concentrations. The method caninclude, after the screening, determining which nucleic acid of themixture was introduced into one or more of the cells.

[0120] In an embodiment, the method includes assaying one or moreproteins of the plurality for a cell-mediated activity, a biochemicalproperty, a structural property, or a physiological property (e.g.,bioavailability).

[0121] The method can further include assaying one or more proteins ofthe plurality in a subject organism.

[0122] The method can also further include selecting one or moreproteins that meet a criterion based on the assaying. The proteins canbe mutagenized, e.g., individually or as an ensemble (e.g., using DNAshuffling or chain shuffling).

[0123] In some implementations, the selected proteins are mutagenized enmasse.

[0124] The expressing can include identifying cells that have at least athreshold level of fusion protein expression. The identifying caninclude FACS, magnetic particle-based sorting, or an automatedselection.

[0125] In one embodiment the identifying includes an automatedscreening.

[0126] In another aspect, the invention features a method ofreformatting hetero-oligomeric receptors for production in mammaliancells. The method (which can be, e.g., a machine-based method) includes:providing a plurality of nucleic acid library members; determining anassessment for each library member with respect to a property; storinginformation about the assessments of the library members in a database;filtering the information to identify a subset of the library members;and reformatting each member of the subset for expression in a mammaliancell by a method that comprises disposing nucleic acid for each memberof the selected subset into a single container. Each of the nucleic acidlibrary members of the plurality, for example, encodes componentpolypeptides of a hetero-oligomeric receptor, e.g., the immunoglobulinheavy chain and immunoglobulin light chain.

[0127] In one embodiment, the library members are formatted, e.g., foryeast display, phage display, or bacterial expression, prior to thereformatting.

[0128] The determining can include contacting a protein array with atarget, a protein corresponding to each library member being present atan address of the array.

[0129] The invention also features a machine-accessible medium thatincludes, encoded thereon or therein data representing (a) identifiersfor library members that each encode a polypeptide, (b) results of afirst functional assays for at least some of the library members; (c)results of a second functional assays for at least some of the librarymembers; and associations that relate the results of the first and/orsecond functional assays and the library member identifiers, wherein thesecond functional assay depends on an activity that is dependent on agiven effector domain (e.g., an Fc domain), and the first functionalassay depends on an activity that is independent of the given effectordomain (e.g., the Fc domain). For example, the second functional assaymay be a cell based assay or an organism-based assay. One cell-basedassay is cell-mediated cytotoxicity.

[0130] The methods described herein can include accessing the medium andfiltering the data for a criterion, e.g., and reformatting one or moreentities based on the filtering.

[0131] The invention also features a nucleic acid that includes: a firstpromoter, a first signal sequence, a first coding region, an interveningsegment, a second coding region, and a third coding region, fused inframe to the second coding region (the second and third coding regionscan be heterologous or not). In one embodiment, the intervening segmentincludes, for example, a eukaryotic promoter, a prokaryotic promoter,and a leader sequence that is cleaved in eukaryotic and prokaryoticcells. In another embodiment, intervening segment further includes anIRES, e.g., upstream and a leader sequence that is cleaved in eukaryoticand prokaryotic cells. See, e.g., upper and lower panels of FIGS. 9 and10.

[0132] The “effector domain” can be any functional domain that canproduce a signal or effect. Non-limiting examples of effector domainsinclude an immunological effector domain, a labelling domain, anenzymatic domain, or a non-immunoglobulin cell-binding domain. Oneexemplary class of effector domains includes effector domains that arefunctional in the extracellular environment. Such domains differ, forexample, from a transcriptional activation domain which function withinthe nucleus of a eukaryotic cell.

[0133] A “localization signal” is a polypeptide sequence that determinesthe subcellular localization of polypeptide or protein. The subcellularlocalization may be: cytoplasmic, nuclear, nuclear envelope,transmembrane, plasma membrane, plasma membrane outer leaflet,endoplasmic reticulum, Golgi, lysosomal, receptor-coated pits, and soforth. For example, the peptide sequence KDEL (SEQ ID NO:32) is anendoplasmic reticulum localization signal and when grafted onto aheterologous protein results in its endoplasmic reticulum localization.An antigen binding protein can be localized within a cell, e.g., asdescribed by Marasco (2001) Curr Top Microbiol Immunol. 260:247-70.

[0134] An exemplary immunological effector domain includes the Fcdomain. The Fc domain binds to host tissues or factors, includingvarious cells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The effector domaincan include an Fc domain or component thereof, e.g., CH2, CH3, CH4,CH2-CH3, or CH2-CH3-CH4. The effector domain can, in someimplementations, include the hinge region, i.e., the region between CH1and CH2. A typical Fc region includes Hinge, CH2 and CH3 and forms adimeric structure. The dimer can optionally include a disulfide bridgeor chemical crosslink.

[0135] The Fc domain can also be of any isotype (for human Fc's, e.g.,IgM, IgG1, IgG2, IgG3, or IgG4). In a preferred embodiment, the Fceffector domain is glycosylated, e.g., at the asparagine correspondingto asparagine 297 of IgG (Kabat numbering). Preferably, the Fc domaincan bind Clq, e.g., if aggregated, and can bind an Fc receptor, e.g.,FCγR1, FCγRIIA, FCγRIIB, FCγRIIIA, or FCγRIIIB. In a much-preferredembodiment, when aggregated, the effector domain elicits a response,e.g., a cytotoxic response, from leukocytes, e.g., NK cells.

[0136] Other effector domains include domains that can produce signals,e.g., green fluorescent protein and derivatives thereof, luciferase,alkaline phosphatase, and horseradish peroxidase. In a preferredembodiment, the effector domain comprises a cytotoxin or cytotoxincomponent, e.g., a chain of diphtheria toxin, ricin, or cholera toxin.

[0137] As used herein, “specific binding” refers to the property of aprotein, e.g., a target or antigen-binding protein or domain: (1) tobind to a target with an affinity of at least 1×10⁷ M⁻¹, and (2) topreferentially bind to the target with an affinity that is at leasttwo-fold greater than its affinity for binding to a non-specific target(e.g., BSA or casein)

[0138] As used herein, the term “antibody” refers to a proteincomprising at least one, and preferably two, heavy (H) chain variableregions (abbreviated herein as VH), and at least one and preferably twolight (L) chain variable regions (abbreviated herein as VL). Thus, theterm encompasses fragments of full-length antibodies (e.g., Fabs) whichhave functional antigen binding properties (see also below), and includeone heavy chain variable region and one light chain variable region.

[0139] The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDR's has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia etal. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein byreference). Each VH and VL is composed of three CDR's and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0140] The antibody can further include a heavy and light chain constantregion, to thereby form a heavy and light immunoglobulin chain,respectively. In one embodiment, the antibody is a tetramer of two heavyimmunoglobulin chains and two light immunoglobulin chains, wherein theheavy and light immunoglobulin chains are inter-connected by, e.g.,disulfide bonds. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. The light chain constant region is comprisedof one domain, CL. The variable region of the heavy and light chainscontains a binding domain that interacts with an antigen. The constantregions of the antibodies typically mediate the binding of the antibodyto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (Clq) of the classicalcomplement system. The term “full-length antibody” includes intactimmunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof), wherein the light chains of the immunoglobulin may be of typeskappa or lambda. As used herein, “isotype” refers to the antibody class(e.g., IgM or IgG1) that is encoded by heavy chain constant regiongenes.

[0141] As used herein, the term “immunoglobulin” refers to a proteinconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes. The recognized human immunoglobulin genes includethe kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3,IgG4), delta, epsilon and mu constant region genes, as well as themyriad immunoglobulin variable region genes. Full-length immunoglobulin“light chains” (about 25 Kd or 214 amino acids) are encoded by avariable region gene at the N-terminus (about 110 amino acids) and akappa or lambda constant region gene at the C-terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids).

[0142] An “immunoglobulin domain” refers to a domain from the variableor constant domain of immunoglobulin molecules. The term “immunoglobulinsuperfamily domain” is distinguished from “immunoglobulin domain.” An“immunoglobulin superfamily domain” refers to a domain that has athree-dimensional structure related to an immunoglobulin domain, but isfrom a non-immunoglobulin molecule. Immunoglobulin domains andimmunoglobulin superfamily domains typically contains two β-sheetsformed of about seven β-strands, and a conserved disulphide bond (see,e.g., Williams and Barclay 1988 Ann. Rev Immunol. 6:381-405). Proteinsthat include immunoglobulin superfamily domains include CD4, plateletderived growth factor receptor (PDGFR), and intercellular adhesionmolecule (ICAM). Immunoglobulin superfamily domains from these proteins,for example, are consider non-immunoglobulin target-binding domains ifthey function to bind a specific target. The term “antigen-bindingfragment” of an antibody (or “antigen binding protein”), as used herein,refers to one or more fragments of a full-length antibody that retainthe ability to specifically bind to a target (e.g., an antigen such apolypeptide or a hapten). Examples of binding fragments encompassedwithin the term “antigen-binding fragment” of an antibody include (i) aFab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding fragment” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

[0143] The “natural N-terminus” of an antibody refers to, for aparticular isotype and species, the naturally occurring N-terminalsequence after processing of the naturally occurring signal sequence(regardless of signal sequence actually used). For example, the naturalN-terminus of a κ light chain is DIQ.

BRIEF DESCRIPTION OF THE DRAWINGS

[0144]FIGS. 1A and 1B are block diagrams depicting exemplary aspects ofa ligand discovery platform.

[0145]FIG. 2 is a reformatting scheme for en masse transfer of Fabs tothe pBRV expression vector. (Top) Organization of display vector:prokaryotic promoter (LacZ), bacterial ribosomal binding site (this onenot shown), M13 geneIII leader, LC coding region (ApaL1/Asc1 fragment),bacterial ribosome binding site (rbs), PelB leader, VH coding sequence(inserted as Sfi1/BstE2 fragment), and M13 geneIII coding sequence(fused in frame).

[0146] (Middle) Fab cassette of the display vector inserted in mammalianexpression vector pBRV as ApaL1/BstE2 fragment. 5′ of LC: HCMV immediateearly promoter and eukaryotic leader sequence, 3′ of VH: constant regionof human IgG1 heavy chain.

[0147] (Bottom) Prokaryotic rbs and leader are removed and eukaryotic“internal ribosome entry site (IRES) and eukaryotic leader sequence areinserted via Asc1 and Asc1 restriction sites.

[0148]FIG. 3 is a schematic of pBRV. The plasmid includes an antibodyexpression cassette, and also the neomycin resistance gene as aselectable marker for generation of stable cell lines; the SV40 originof replication. pRRV is similar to pBRV, except for the precise positionof the ApaL1 site in the geneIII leader adjacent to VL (after Fabinsertion).

[0149]FIG. 4 is a schematic of regulatory elements (IRES+leadersequence: R27080 leader is SEQ ID NO: 1; Ab leader is SEQ ID NO:2)located between the first and second coding regions in pblue and pRRV(shuttle vector).

[0150]FIG. 5 is a reformatting scheme for transfer of individual Fabs topRRV.

[0151] (Top) Organization of display vector: prokaryotic promoter(LacZ), M13 geneIII leader, complete LC (ApaL1/Asc1 fragment), bacterialribosome binding site (rbs), PelB leader, VH (inserted as Sfi1/BstE2fragment) CH1 fused to M13 geneIII.

[0152] PCR primer binding sites are shown as arrows. The forward primeranneals to the 5′end of the V gene. The reverse primer binds in CH1. Thecomplete Fab insert is amplified by PCR.

[0153] (Middle) Fab cassette of the display vector inserted in mammalianexpression vector pBRV as ApaL1/BstE2 fragment. 5′ of LC: HCMV immediateearly promoter and eukaryotic leader sequence, 3′ of VH: constant regionof human IgG1 heavy chain.

[0154] (Bottom) Prokaryotic rbs and leader are exchanged againsteukaryotic “internal ribosome entry site (IRES) and eukaryotic leadersequence via Asc1 and Mfe 1.

[0155]FIG. 6 is a schematic of pRRV, including a complete antibodyexpression cassette, after reformatting

[0156]FIG. 7 is a schematic of a regulatory element consisting of polyAsite, a second eukaryotic promoter and a leader sequence functional inmammalian cells.

[0157] This set-up, introduction of a second promoter to drive HCexpression, can be used instead of IRES.

[0158]FIG. 8 depicts leader sequence preferences.

[0159]FIG. 9 is a schematic of an exemplary integrated expressionvector. A Fab expression cassette in the vector can be shuttled betweena prokaryotic Fab display system (top) and a mammalian IgG expressionsystem (bottom). The Fab cassette is transferred as an ApaL1/Nhe1fragment, in a single cloning step. Since, for the heavy chain, a leaderis used that is functional in bacteria and mammalian cells, andregulatory elements for pro- and eukaryotic expression are supplied, nofurther (i.e. second) cloning steps are required.

[0160] (Top)—Fab display construct: a prokaryotic bicistronic expressioncassette consisting of the following elements: P_(B), prokaryoticpromoter (e.g. LacZ); L_(B), bacterial leader; VL-CL, light chainportion of Fab; IRES, internal ribosome entry; P_(B), prokaryoticpromoter; L_(E,B), bifunctional leader (functional in bacteria and inmammalian cells); VH-CH1, heavy chain portion of Fab; III, gene III of afilamentous phage.

[0161] (Bottom)—IgG expression cassette: P_(E), eukaryotic promoter;L_(E), eukaryotic leader; VL-CL, light chain of antibody; IRES, internalribosome entry site; P_(B), prokaryotic promoter; L_(E,B), bifunctionalleader (functional in bacteria and in mammalian cells);VH-CH1-H-CH2-CH3, antibody heavy chain regions; pA, poly-adenylationsite.

[0162]FIG. 10 is a schematic of another exemplary integrated expressionvector. A Fab expression cassette in the vector can be shuttled betweena prokaryotic Fab display system (top) and a mammalian IgG expressionsystem (bottom). The Fab cassette is transferred as an ApaL1/Nhe1fragment, in a single cloning step. Since, for the heavy chain, a leaderis used that is functional in bacteria and mammalian cells, andregulatory elements for pro- and eukaryotic expression are supplied, nofurther (i.e. second) cloning steps are required.

[0163] (Top)—Fab display construct: a prokaryotic bicistronic expressioncassette consisting of the following elements: P_(B), prokaryoticpromoter (e.g. LacZ); L_(B), bacterial leader; VL-CL, light chainportion of Fab; pA, eukaryotic poly-adenylation signal and P_(E),eukaryotic promoter (e.g. HCMV IE promoter; P_(B), prokaryotic promoter;L_(E,B), bifunctional leader (functional in bacteria and in mammaliancells); VH-CH 1, heavy chain portion of Fab; III, gene III of afilamentous phage.

[0164] (Bottom)—IgG expression cassette: P_(E), eukaryotic promoter;L_(E), eukaryotic leader; VL-CL, light chain of antibody; pA, eukaryoticpoly-adenylation signal and P_(E), eukaryotic promoter (e.g. HCMV IEpromoter e; P_(B), prokaryotic promoter; L_(E,B), bifunctional leader(functional in bacteria and in mammalian cells); VH-CH1-H-CH2-CH3,antibody heavy chain regions; pA, poly-adenylation site.

[0165]FIG. 11 is an example of a FACS selection of antibody expressingcells which capture the secreted antibody at their surface. Cells withhighest expression levels (upper X%) can be isolated.

[0166]FIG. 12 is a schematic of procedure of cell surface attachment anddetection of a secreted antibody on an expressing cell.

[0167] The procedure depicted consists of: surface biotinylation ofantibody expressing cells, attachment of streptavidin conjugatedcapturing reagent (e.g. HC specific antibody), capturing of secretedantibody at the cell surface of the expressing cell and detection ofsurface associated secreted soluble antibody with fluorescent dyeconjugate (e.g. anti LC F(a,b)₂-FITC).

[0168]FIG. 13 is a schematic of the use of a low permeability mediumduring the FACS staining process. Secreting and non-secreting cells aresurface biotinylated, and an antibody-capturing matrix is applied toboth types of cells. Culture of cells in “medium of low permeability”during the antibody capturing phase protects cross-feeding ofnon-expressing by antibody expressing cells.

[0169]FIG. 14 is a schematic of an information management system.

[0170]FIG. 15 is a flow chart.

[0171]FIG. 16 is a schematic of an automated screening system.

[0172]FIG. 17 depicts reformatting Fab's in a phage display vector fordisplay on yeast cells by a yeast display vector. Combinations of Fabheavy and light chains are transferred into a yeast display vector inwhich expression is under control of the GAL1 promoter. The reformattingyields a yeast display vector that supports VH-CH1 expression as an Aga2fusion protein. In the region between the two Fab chains, the followingelements are provided: a second copy of GALL promoter, a yeast leadersequence (ss) and Aga2p coding segment that encodes a domain fused tothe VH N-terminus.

[0173]FIGS. 18, 19, 20, and 21 are exemplary reformatting schemes.

[0174]FIGS. 22A and 22B provide a map and translation of IgG1 HCconstant region fragment as found in pBRV and pRRV.

[0175]FIG. 23 is a map of pRRV.

[0176]FIG. 24 is a map of pBRV.

[0177]FIG. 25 is a map of pBlueIRES.Mfe. The top nucleic acid sequenceis SEQ ID NO:17, its complement (middle) is SEQ ID NO:18; the encodeamino acid sequence is SEQ ID NO:19.

[0178]FIG. 26 is a map of pBLUE.IRES.Sfi. The top nucleic acid sequenceis SEQ ID NO:20, its complement (middle) is SEQ ID NO:21; the encodeamino acid sequence is SEQ ID NO:22.

[0179]FIG. 27 is a map of pShuttleI.

[0180]FIG. 28 is a map of pShuttleII

[0181]FIG. 29 is a map of pShuttleIII.

DETAILED DESCRIPTION

[0182] The invention provides, in part, platforms for identifyingligands that recognize targets. The ligands can be, for example,antibodies, T cell receptors, and immunoadhesin-type molecules. Many ofthe ligands include or can be linked to an effector domain.

[0183] In one exemplary aspect, a platform for antibody discovery isdescribed. The platform, of course, can be adapted for theidentification of other ligands, including those described hereinafter.

[0184] Referring now to FIG. 1, the platform includes one or moreantibody libraries (10). A library, e.g., a display library, can bescreened to identify members that bind to a target. A variety ofselection methods are available (20). In addition, targets can beselected from a target collection (30).

[0185] In some embodiments, members of the antibody library are screenedagainst multiple targets in parallel. After selection and optionalprocessing steps, the target bound by each member can be determined.Examples of multiple targets include a cell that displays heterogeneousepitopes, a population of cells, a cell extract, polypeptides expressedby a cDNA library or cell-specific library, and so forth.

[0186] After one or more rounds of selections (e.g., two rounds ofselections), individual candidate antibodies are isolated from theselected fraction of the library. Automated methods of screening (40)each individual candidate are described herein. Candidates which satisfycertain criteria during the screening process are reformatted (50) andadvanced to IgG production (60). In another case, the selectedrepertoire of individual clones is reformatted provided a certainfraction of the clones satisfy a certain criterion in the screeningprocess. This application provides a number of exemplary reformattingmethods for efficiently transferring nucleic acids encoding eachcandidate from the initial library vector into a vector for eukaryoticexpression. As seen below, the transfer process can be implemented enmasse or candidate-by-candidate. Once transferred to the eukaryoticexpression vector, the nucleic acid is introduced into the eukaryoticcells (typically mammalian) and expressed.

[0187] The transfer process can be implemented on a variety of scales.In one example, to produce a monoclonal immunoglobulin, an individualnucleic acid encoding a mono-specific ligand is reformatted. In anotherexample, multiple nucleic acids (e.g., each encoding a differentmono-specific ligand that binds to a different epitope of the sametarget molecule) are reformatted as Ig and introduced into cells asmonoclonal DNA (e.g., individual species) preparations, oligoclonal DNAensembles (e.g., a defined set) or polyclonal DNA ensembles (e.g., anincompletely characterized set of uncertain number of individual clones)for protein production. Whole IgG (or other isotypes/variants)antibodies can be isolated from the cells or media conditions by theexpressing cells.

[0188] In the case of cells transfected with oligoclonal or polyclonalDNA ensembles, additional diversity may arise additional species if morethan one DNA species enters an individual. This degree diversity can becontrolled, e.g., by varying the nucleic acid concentration and/ortransfection conditions so that cells only take up a single DNAmolecule. Added diversity is useful in some embodiments, and undesirablein others.

[0189] The reformatted antibody genes can be introduced into eukaryoticcells for production of IgG by a variety of transfection methods. Theproduction of IgG can use a transient system in which the Ig isharvested from the supernatant of cultures containing cells into whichthe reformatted DNA is introduced. In such transient systems, noselection to find cells that have integrated the antibody genes intotheir chromosomes is required. Special vector elements can be providewithin the eukaryotic expression vector such as the SV40 origin ofreplication. After introduction of such vector in cells that harbor thelarge T antigen of SV40 (such as certain COS cell lines), the DNA isamplified as an episome, leading to a higher level of transientexpression of the Ig. Other cells that can be used for expression areChinese Hamster Ovary (CHO) cells, African green monkey (COS) cells,Human Embryonic Kidney 293 T (Hek293 T) cells, etc. If selection markersare provide for in the expression vector, cells can be selected aftertransfection and clones identified that stably express the IgG. IgGantibodies are isolated from the cells or media.

[0190] The IgG antibodies can then be assayed for a cellular activity(70), cell binding (80), and biochemical activity (90). Information forthe performance of each antibody in these assays can be stored in adatabase. Candidate antibodies can be selected based on the collecteddata. The best candidates may be subjected to an in vitro antibodymaturation process (100). Variants of the best candidates re-enter theprocess at the level of a second selection (20), screening (40), or evenIgG production (60). Transient transfection of cells can be sufficientto produce some quantities of antibodies. In other circumstances, e.g.,for larger quantities, stable cell lines that include DNA encoding thereformatted IgG integrated into their genome, are used.

[0191] Antibodies identified by the above process can be used in in vivoassays (e.g., using animal models) and, generally, as preclinicaltargets 110.

[0192] Further exemplary implementation details for aspects of thisantibody development platform and related platforms are provided below.

[0193] Antibody Libraries

[0194] A nucleic acid library whose members encode antibodies, e.g.,Fabs can be constructed by the exemplary methods described in de Haardet al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998)Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8and references described therein.

[0195] In one embodiment, an antibody display library of Fabs isconstructed in a phage display vector. Exemplary vectors include anexpression cassette that encodes a bicistronic transcript having regionsfor the expression of the antibody light and heavy chain. Thesecassettes can include the following elements: (1) a promoter suited forconstitutive or inducible expression (e.g., lac promoter); (2) aribosome binding site and signal sequence preceding the light and heavychain cloning regions; (3) optionally, a region following the heavy orlight chain cloning region that encodes a tag sequence such as a stretchof 5-6 histidines or an epitope recognized by an antibody; (4) asuppressible codon (e.g., an amber codon); and (5) a sequence encoding aphage coat protein positioned in-frame to form a fusion to the 3′ end ofeither the heavy or light chain.

[0196] With respect to an exemplary phagemid system, genes encoding VHand VL regions are cloned into the vector as follows. Variable heavychain region genes are cloned as VH-gene fragments. The vector suppliesall Fab's with a human CH1 domain. The VH-CH1 encoding sequence formedby insertion of the VH-gene fragments to the vector is fused (in thevector) to a sequence encoding tags for purification and detection ahistidine tail for Immobilized Metal Affinity Chromatography (Hochuli,et al., (1988) BioTechnology 6:1321-132S) and a c-myc-derived tag(Munro, et al. (1986) Cell 46:291-300)), followed by an amber stop codon(Hoogenboom, et al. (1991) Nucleic Acids Res. 19:4133-4137) and theminor coat protein III of filamentous phage fd. The antibody light chainis cloned as full VL-CL fragment, for directed secretion and assemblywith the VH-CH1 on the phage particle. When the bicistronic mRNA istranscribed and translated in a amber-suppressing host cell, bothpolypeptide subunits are produced and join in the periplasm to produceFab fragments that are tethered to the phage gene III protein orfragment thereof.

[0197] Antibody libraries can be incorporate diversity from a variety ofsources, including from synthetic nucleic acid, naive nucleic acids,patients (e.g., immunized or diseased human subjects), and animals(e.g., immunized animals).

[0198] Natural Immune Sources. In one embodiment, immune cells can beused as a natural source of diversity for the variation of antibodies,MHC-complexes and T cell receptors. Some examples of immune cells are Bcells and T cells. The immune cells can be obtained from, e.g., a human,a primate, mouse, rabbit, camel, or rodent. The cells can be selectedfor a particular property. For example, T cells that are CD4+ andCD8-can be selected. B cells at various stages of maturity can beselected.

[0199] In another embodiment, fluorescent-activated cell sorting is usedto sort B cells that express surface-associated IgG molecules. Further Bcells expressing different isotypes of IgG can be isolated. In anotherembodiment, the B or T cell is cultured in vitro. The cells can bestimulated in vitro, e.g., by culturing with feeder cells or by addingmitogens or other modulatory reagents, such as antibodies to CD40, CD40ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide,concanavalin A, phytohemagglutinin or pokeweed mitogen.

[0200] In still another embodiment, the cells are isolated from asubject that has an immunological disorder, e.g., systemic lupuserythematosus (SLE), rheumatoid arthritis, vasculitis, Sjogren'ssyndrome, systemic sclerosis, or anti-phospholipid syndrome. The subjectcan be a human, or an animal, e.g., an animal model for the humandisease, or an animal having an analogous disorder. In still anotherembodiment, the cells are isolated from a transgenic non-human animalthat includes a human immunoglobulin locus.

[0201] In one embodiment, the cells have activated a program of somatichypermutation. Cells can be stimulated to undergo somatic mutagenesis ofimmunoglobulin genes, for example, by treatment withanti-immunoglobulin, anti-CD40, and anti-CD38 antibodies (see, e.g.,Bergthorsdottir et al. (2001) J Immunol. 166:2228). In anotherembodiment, the cells are naive.

[0202] Naturally diverse sequences can be obtained as cDNA produced frommRNAs isolated from cell and samples described herein. Full length(i.e., capped) mRNAs are separated (e.g. by degrading uncapped RNAs withcalf intestinal phosphatase). The cap is then removed with tobacco acidpyrophosphatase and reverse transcription is used to produce the cDNAs.The reverse transcription of the first (antisense) strand can be done inany manner with any suitable primer. See, e.g., de Haard et al. (1999)J. Biol. Chem 274:18218-30. The primer binding region can be constantamong different immunoglobulins, e.g., in order to reverse transcribedifferent isotypes of immunoglobulin. The primer binding region can alsobe specific to a particular isotype of immunoglobulin. Typically, theprimer is specific for a region that is 3′ to a sequence encoding atleast one CDR. Poly-dT primers (e.g., for the heavy-chain genes) orsynthetic primers that hybridize to a synthetic sequence ligated to themRNA strand may also be used.

[0203] cDNA can be amplified, modified, fragmented, or cloned into avector to form an antibody library. See, e.g., de Haard et al. (1999)supra. Also, for example, see U.S. Provisional Application No.60/343,954, filed Oct. 24, 2001, “HYBRIDIZATION CONTROL OF SEQUENCEVARIATION” describes a method of cleaving cDNA usingoligonucleotide-directed cleavage and incorporating immunologicaldiversity into a template immunoglobulin sequence.

[0204] Murine-Derived Human Immunoglobulins. In one embodiment, theimmunize animal is a transgenic animal (e.g., a mouse) that has humanimmunoglobulin genes. See, e.g., U.S. Pat. No. 6,150,584; Fishwild etal. (1996) Nature Biotechnol. 14:845-85; Mendez et al. (1997) NatureGenet. 15:146-156; Nicholson et al. (1999) J. Immunol. 163:6898. Onesuch transgenic mouse can be constructed as described in WO 94/02602using a YAC for the human heavy chain locus, e.g., yHIC (1020 kb), andhuman light chain locus YAC, e.g., yK2 (880 kb). yHIC includes 870 kb ofthe human variable region, the entire D and JH region, human μ, δ, γ2constant regions and the mouse 3′ enhancer. yK2 includes 650 kb of thehuman kappa chain proximal variable region (Vκ), the entire Jκ region,and Cκ with its flanking sequences. Administration of an antigen to suchmice elicits the generation of human antibodies against the antigen. Thespleens of such mice are isolated. mRNA encoding the human antibodygenes is extracted and used to produce a nucleic acid library encodingantibodies against the antigen. In some implementations, the library ismutagenized, e.g., affinity matured, in vitro prior to selection andscreening.

[0205] Synthetic Diversity. A nucleic acid library can also includesynthetic diversity at one or more positions, e.g., in one or more CDRs.Libraries can include regions of diverse nucleic acid sequence thatoriginate from artificially synthesized sequences. Typically, these areformed from degenerate oligonucleotide populations that include adistribution of nucleotides at each given position. The inclusion of agiven sequence is random with respect to the distribution. One exampleof a degenerate source of synthetic diversity is an oligonucleotide thatincludes NNN wherein N is any of the four nucleotides in equalproportion.

[0206] Synthetic diversity can also be more constrained, e.g., to limitthe number of codons in a nucleic acid sequence at a given trinucleotideto a distribution that is smaller than NNN. For example, such adistribution can be constructed using less than four nucleotides at somepositions of the codon. In addition, trinucleotide addition technologycan be used to further constrain the distribution.

[0207] So-called “trinucleotide addition technology” is described, e.g.,in Wells et al. (1985) Gene 34:315-323, U.S. Pat. No. 4,760,025 and WO91/19818. Oligonucleotides are synthesized on a solid phase support, onecodon (i.e., trinucleotide) at a time. The support includes manyfunctional groups for synthesis such that many oligonucleotides aresynthesized in parallel. The support is first exposed to a solutioncontaining a mixture of the set of codons for the first position. Theunit is protected so additional units are not added. The solutioncontaining the first mixture is washed away and the solid support isdeprotected so a second mixture containing a set of codons for a secondposition can be added to the attached first unit. The process isiterated to sequentially assemble multiple codons. Trinucleotideaddition technology enables the synthesis of a nucleic acid that at agiven position can encoded a number of amino acids. The frequency ofthese amino acids can be regulated by the proportion of codons in themixture. Further the choice of amino acids at the given position is notrestricted to quadrants of the codon table as is the case if mixtures ofsingle nucleotides are added during the synthesis.

[0208] Selections

[0209] The selection process (20) can be performed manually or using anautomated method. In some cases, non-specific binding and othernon-ideal properties require more than one selection cycle. Additionalselection cycles increase the enrichment for candidate library members.To repeat a selection step, eluted library members are amplified thenreapplied to the target ligand. Depending on the implementation,different numbers of selection cycles may be sufficient to identify apool of candidate library members from a library having a vastdiversity. For example, one, or two rounds of selection may besufficient. A set of selection cycles is referred to as a selectioncampaign.

[0210] Some exemplary selection processes are as follows.

[0211] Panning. The target molecule is immobilized to a solid supportsuch as a surface of a microtitre well, matrix, particle, or bead. Thedisplay library is contacted to the support. Library members that haveaffinity for the target are allowed to bind. Non-specifically or weaklybound members are washed from the support. Then the bound librarymembers are recovered (e.g., by elution) from the support. Recoveredlibrary members are collected for further analysis (e.g., screening) orpooled for an additional round of selection.

[0212] Magnetic Particle Processor. One example of an automatedselection uses magnetic particles and a magnetic particle processor. Inthis case, the target is immobilized on the magnetic particles, e.g., asdescribed below. The KingFisher™ system, a magnetic particle processorfrom Thermo LabSystems (Helsinki, Finland), is used to select displaylibrary members against the target. The display library is contacted tothe magnetic particles in a tube. The beads and library are mixed. Thena magnetic pin, covered by a disposable sheath, retrieves the magneticparticles and transfers them to another tube that includes a washsolution. The particles are mixed with the wash solution. In thismanner, the magnetic particle processor can be used to serially transferthe magnetic particles to multiple tubes to wash non-specifically orweakly bound library members from the particles. After washing, theparticles are transferred to a tube that includes an elution buffer toremove specifically and/or strongly bound library members from theparticles. These eluted library members are then individually isolatedfor analysis (e.g., screening) or pooled for an additional round ofselection.

[0213] An exemplary magnetically responsive particle is the Dynabead®available from Dynal Biotech (Oslo, Norway). Particles can be blockedwith a blocking agent, such as serum albumin (e.g., BSA) or casein toreduce non-specific binding and coupling of compounds other than thetarget to the particle. The target is attached to the paramagneticparticle directly or indirectly. A variety of target molecules can bepurchased in a form linked to paramagnetic particles. In one example, atarget is chemically coupled to a particle that includes a reactivegroup, e.g., a crosslinker (e.g., N-hydroxy-succinimidyl ester) or athiol.

[0214] In another example, the target is linked to the particle using amember of a specific binding pair. For example, the target can becoupled to biotin. The target is then bound to paramagnetic particlesthat are coated with streptavidin (e.g., M-270 and M-280 StreptavidinDynaparticles® available from Dynal Biotech, Oslo, Norway). In oneembodiment, the target is contacted to the sample prior to attachment ofthe target to the paramagnetic particles. Other specific binding pairs(e.g., a peptide epitope and corresponding monoclonal antibody) can beused. (see, e.g., Kolodziej and Young (1991) Methods Enz. 194:508-519).

[0215] Capillary Device for Washing Magnetic Beads. Provisionalapplication No. 60/337,755, filed Dec. 7, 2001, “Method and Apparatusfor Washing Magnetically Responsive Particles” describes an apparatusand methods that can, in one implementation, be used to wash magneticparticles in a capillary tube. On exemplary apparatus features acapillary that houses magnetic particles. The chamber is located betweena first magnet and a second magnet. The magnets and are attached to aframe that can be actuated from a first position to a second position.When the frame is actuated, the magnetic particles in the capillary areagitated.

[0216] To use the apparatus for display library screening, librarymembers are contacted to magnetic particles that have an attachedtarget. The particles are disposed in the capillary (before, during, orafter the contacting). Then, the particles are washed in the capillarywith cycles of agitation and liquid flow to remove non-specifically orweakly bound library members. After washing, bound library members canbe eluted or dissociated from the particles and recovered.

[0217] MiniMACS™ is still another device, available from Miltenyi BiotecGmbH (Gladbach, Germany), which can be used for magnetic particle-basedseparations.

[0218] Cell-Based Selections. The selection can be performed by bindingthe display library to target cells, and then selecting for librarymembers that are bound by the cells. Cell-based selections enable theidentification of ligands that recognize target molecules as presentedin their natural milieu, e.g., including post-translationalmodifications, associated proteins and factors, and competing factors.Further, since cell-based selections are not directed against a specificsingular target molecule, no a priori information is required about thetarget. Rather, the cell itself is a determinant. Later steps,particular functional assays, can be used to verify that identifiedligands are active in targeting effector functions to the cell.

[0219] In one embodiment, the selection further requires that thelibrary members are internalized by the target cells. This methodselects both for binding of the antibody ligand to the target cell andfor endocytosis of the antibody ligand into the cell. See, e.g., Heitneret al. (2001) J. Immunol. Methods. 248:17-30; Poul et al. (2000) J. Mol.Biol. 301:1149-1161.

[0220] Non-limiting examples of cells include cancer cells,hematopoietic cells, fibroblasts, transformed cells, BalI cells, and soforth. Such cells are attached to magnetically responsive particlesusing an antibody specific for a marker on the cell surface, e.g., CD19or a cell-surface cancer-specific antigen (for example, hypoglycosylatedMUC1, melanoma differentiation antigen gp100, or CEA1.).

[0221] Another class of targets includes cells, e.g., fixed or livingcells. The cell can be bound to an antibody that is attached to a solidsupport (e.g., a paramagnetic particle or a surface of a growthchamber). For example, a biotinylated rabbit anti-mouse Ig antibody isbound to streptavidin paramagnetic beads and a mouse antibody specificfor a cell surface protein of interest is bound to the rabbit antibody.

[0222] In one embodiment, the cell is a recombinant cell, e.g., a celltransformed with a heterologous nucleic acid that expresses aheterologous gene or that disrupts or alters expression of an endogenousgene. The cells can be transformed with a plasmid that expresses (e.g.,under control of an inducible or constitutive promoter) a cell-surfaceprotein of interest. The plasmid can also express a marker protein,e.g., for use in binding the transformed cell to a solid support, e.g.,a magnetic particle. The cells can express a heterologous intracellularprotein, e.g., an oncogene, transcription factor, or cell-signallingprotein. The intracellular protein can alter cell behavior or therepertoire of molecules on the cell surface.

[0223] In another embodiment, the cell is a primary culture cellisolated from a subject, e.g., a patient, e.g., a cancer patient. Instill another embodiment, the cell is a transformed cell, e.g., amammalian cell with a cell proliferative disorder, e.g., a neoplasticdisorder. In still another embodiment, the cell is the cell of apathogen, e.g., a microorganism such as a pathogenic bacterium,pathogenic fungus, or a pathogenic protist (e.g., a Plasmodium cell) ora cell derived from a multicellular pathogen.

[0224] Cells can be treated, e.g., at a particular stage of theselection. The treatment can be a drug or an inducer of a heterologouspromoter-subject gene construct. The treatment can cause a change incell behavior, morphology, and so forth. In some implementations,display library members that associate or dissociate from the cells upontreatment are collected and analyzed.

[0225] In still another embodiment, the cells are treated (e.g., using adrug or genetic alteration) to alter the rate of endocytosis,pinocytosis, exocytosis, and/or cell secretion.

[0226] In vivo Selections. The selection can be done in vivo to identifylibrary members that bind to a target tissue or organ, e.g., asdescribed in Kolonin et al. (2001) Current Opinion in Chemical Biology5:308-313, Pasqualini and Ruoslahti (1996) Nature 380:364-366, andPaqualini et al. (2000) “In vivo Selection of Phage-Display Libraries”In Phage Display: A Laboratory Manual Ed. Barbas et al. Cold SpringHarbor Press 22.1-22.24. For example, a phage display library isinjected into a subject (e.g., a human or other mammal). After anappropriate interval, a target tissue or organ is removed from thesubject and the display library members that bind to the target site arerecovered and characterized.

[0227] Screening

[0228] After selection, the identified members of the pool areindividually isolated and screened. Screening can also be automated (seebelow).

[0229] A screening differs from a selection in that, a screen ischaracterized by the analysis of library members individually (or inpools) whereas a selection is characterized by analysis of librarymembers that are separated from other members during the process (e.g.,retained, eluted, or washed off). In some implementations, a collectionof library members is directly screened, without being subjected to aselection step. This approach, for example, can be used during affinitymaturation and refinement protocols.

[0230] For the screen, each identified member is assayed for afunctional property, typically a binding activity (including, forexample, information related to specificity, a kinetic parameter, anequilibrium parameter, avidity, affinity, and so forth). The functionalinformation can also relate to one or more of the following: astructural or biochemical property (e.g., thermal stability,oligomerization state, solubility and so forth), a physiologicalproperty (e.g., renal clearance, toxicity, target tissue specificity,and so forth) and so forth.

[0231] This information can be obtained in two phases of screening. Inthe primary phase, typically the binding properties of selected displaylibrary members are verified using the binding moiety without aneffector domain. Members that meet a set of selection criteria (or acriterion) are selected for reformatting and mammalian cell expressionwith a linked effector domain. In the secondary phase, functionalinformation is obtained from the mammalian cell-expressed ligandcomplete with a linked effector domain.

[0232] The following describes possible embodiments of exemplary assaysfor binding assays:

[0233] ELISA. In one implementation, ELISA assays are used to evaluatethe affinity of each identified library member for the target and for anon-target. For example, each polypeptide from the identified members iscontacted to a microtitre plate whose bottom surface has been coatedwith the target, e.g., a limiting amount of the target. The plate iswashed with buffer to remove non-specifically bound polypeptides. Thenthe amount of the polypeptide bound to the plate is determined byprobing the plate with an antibody that can recognize the polypeptide,e.g., a tag or constant portion of the polypeptide. The antibody islinked to an enzyme such as alkaline phosphatase, which produces acalorimetric product when appropriate substrates are provided. Thepolypeptide can be purified from cells or assayed in a display libraryformat, e.g., as a fusion to a filamentous bacteriophage coat. Inanother version of the ELISA assay, each polypeptide of a library isused to coat a different well of a microtitre plate. The ELISA thenproceeds using a constant target molecule to query each well.

[0234] Homogeneous Binding Assays. Protein ligands identified from thedisplay library can be screened using a homogenous assay, i.e., afterall components of the assay are added, additional fluid manipulationsare not required. For example, fluorescence resonance energy transfer(FRET) can be used as a homogenous assay (see, for example, Lakowicz etal., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). Another example of a homogenous assay is Alpha Screen(Packard Bioscience, Meriden Conn.). Alpha Screen uses two labeledbeads. One bead generates singlet oxygen when excited by a laser. Theother bead generates a light signal when singlet oxygen diffuses fromthe first bead and collides with it. The signal is only generated whenthe two beads are in proximity. One bead can be attached to the displaylibrary member, the other to the target. Signals are measured todetermine the extent of binding. The homogenous assays can be performedwhile the candidate polypeptide is attached to the display libraryvehicle, e.g., a bacteriophage.

[0235] Protein Arrays. Protein ligands identified from the displaylibrary can also be screened using protein arrays. The polypeptides areimmobilized on a solid support, for example, on a bead or an array. Fora protein array, each of the polypeptides is immobilized at a uniqueaddress on a support. Typically, the address is a two-dimensionaladdress. Methods of producing polypeptide arrays are described, e.g., inDe Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et al.(1999) Anal. Biochem. 270:103-111; Ge (2000) Nucleic Acids Res. 28, e3,I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763; WO 01/40803and WO 99/51773. Polypeptides for the array can be spotted at highspeed, e.g., using commercially available robotic apparati, e.g., fromGenetic MicroSystems or BioRobotics.

[0236] Library members that are specific for the target can becharacterized by nucleic acid sequencing. Sequence information is usedto classify the members and to remove redundant members (i.e., membersthat encode that same antibodies).

[0237] Library members that meet a given criterion (or criteria) areadvanced to IgG Production (50). First, the library members arereformatted from the prokaryotic phage library vector into a vector formammalian cell expression.

[0238] Reformatting

[0239] The reformatting process is used, for example, to transfernucleic acid from a display vector to a vector suitable for mammaliancell production. The transfer process can also include modifications(e.g., substitution, insertion, or deletion of sequences) thatfacilitate expression in a mammalian expression system. Suchmodifications include sequences for transcription, translation,secretion, effector domains function, and selection. Further, othermodifications are also possible, e.g., the inclusion of introns and soforth.

[0240] In one embodiment, each selected library member is reformattedindividually. In another embodiment, the library members are combinedand reformatted en masse.

[0241] The reformatting process can be tailored to the expression systemused initially for display and for the secondary expression system. Forexample, for phage display, the coding of heavy and light chains by asingle bicistronic transcript favors stoichiometric production of thetwo chains as well as efficient transcription and translation. Thisdesign requires a tandem organization of the nucleic acid sequencesencoding the heavy and light chains such that they are in the sametranslational orientation. The reformatting process maintains thisorientation while modifying the sequences to either encode a bicistronictranscript that can be expressed in mammalian cells or to produce twoseparate transcripts. Only two cycles of restriction digestion andligation are required for the reformatting.

[0242] In one example of en masse reformatting, the first cycle includesdigesting display vectors to release nucleic acid fragments that includeminimally a light chain variable coding region and a heavy chainvariable coding region. The fragments are cloned into a vector formammalian expression. During this cycle, the transfer of the nucleicacid fragments encoding both chains insures that combinations of heavyand light chain present in the display vector are maintained in themammalian vector. Further, the transfer process can be used to switchfrom a prokaryotic promoter to a mammalian promoter on the 5′ end of thecoding strand and from a sequence encoding a bacteriophage coat protein(or fragment thereof) to a sequence encoding an Fc domain on the 3′ endof the coding strand. General methods for cloning are described instandard laboratory manuals, e.g., Sambrook et al. (2001) MolecularCloning: A Laboratory Manual (Third Edition), Cold Spring HarborLaboratory Press.

[0243] In the second cycle, the region intervening between the lightchain coding region and the heavy chain-coding region is substituted. Asequence that includes a prokaryotic ribosome binding site is removed,and a sequence with an internal ribosomal entry site (IRES) or asequence including a eukaryotic promoter is inserted. Also in thisprocess the prokaryotic signals for secretion (e.g., a signal sequenceat the 5′ end of the Ig coding region) can be replaced by an eukaryoticsignal sequence. In some implementations, the intervening region issubstituted by recombination in a cell. In still others, the interveningregion is not substitute, but rather sequences are inserted e.g., usingsite-specific recombination, and optionally without excising thesequences designed for prokaryotic expression.

[0244] Hybrid signal sequences that are functional in both prokaryoticand eukaryotic cells can be used to obviate reformatting of some (e.g.,at least the 3′ region of the signal sequence, e.g., the −3, −2, and −1positions) or all of the signal sequence. In some cases, a signalsequence is functional in multiple expression systems (e.g., both pro-and eukaryotic systems). For example, the signal sequence of somebacterial beta-lactamases is functional in eukaryotic cells andprokaryotic cells. See, e.g., Kronenberg et al., 1983, J. Cell Biol. 96,1117-9; Al-Qahtani et al., 1998, Biochem. J. 331, 521-529. Signalsequences that function in multiple hosts can also be designed on thebasis of the requirement of such signal sequence (consensus rules) inthe respective expression hosts (e.g., as shown in FIG. 8), or may beselected empirically.

[0245] Signal sequences that function in both expression systems (e.g.,prokaryotic and eukaryotic) do not need to be replaced by thereformatting procedure. Thus, only one modification may be required. InFIGS. 9 and 10 (top), a vector system is used that requires only onemodification in order to be appropriately reformatted for expression inmultiple expression systems (e.g., prokaryotic and eukaryotic). In thiscase the signal is used to drive the secretion of a heavy chain VH-CH1fused to a phage derived pIII protein, in prokaryotic cells, for Fabphage display, and, after a single recloning step, the expressioncassette encodes a full IgG (i.e., including an Fc region) that can beexpressed in a mammalian cell.

[0246] Features of a Mammalian Expression Vector.

[0247] Some key features of a mammalian expression vector which may beintroduced by the reformatting process include: a transcriptionalregulatory sequence, a internal ribosome entry site, a chromatin controlsequence, a localization signal, and a leader sequences. Similarconsiderations may apply to enhancers, untranslated regions, polyadenylation sites, selectable markers, and so on.

[0248] Transcriptional Regulatory Sequences. Transcriptional controlsequences are used to drive expression of transcripts encoding bothsubunits of the antibody ligand. For high level expression, for example,exemplary enhancer/promoter regulatory elements include elements derivedfrom SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element. See, e.g., U.S. Pat. No. 5,385,839.

[0249] Still other transcriptional regulatory sequences are selected fordriving cell or tissue-specific expression. For example, to express anantibody ligand ectopically in a cytotoxic T cell, a T-cell specificpromoter is used.

[0250] Chromatin Control Sequences. These sequences include elements,e.g., those variously termed insulators (or insulator element), locuscontrol regions (which are frequently tissue specific), and chromatinopening elements (which frequently are not tissue specific). As definedherein, chromatin control sequences are sequences that insulate thetranscription of genes placed within its range of action but which doesnot perturb gene expression, either negatively or positively. Forexample, they modulate (e.g., shield) the regulatory effects ofchromatin and nearby sequences in a nuclear environment, typically achromosomal environment. Thus, insulators can enable sustained and/orappropriate regulatory control of sequences integrated into heterologousregions of a chromosome. Chromatin opening elements can protect a genefrom gene “silencing” mechanisms.

[0251] An insulator sequence can be positioned on either side of the DNAsequence to be transcribed. For example, the insulator can be positionedabout 200 bp to about 1 kb, 5′ from the promoter, and at least about 1kb to 5 kb from the promoter, at the 3′ end of the gene of interest. Inaddition, more than one insulator sequence can be positioned 5′ from thepromoter or at the 3′ end of the transgene. For example, two or moreinsulator sequences can be positioned 5′ from the promoter. Theinsulator or insulators at the 3′ end of the transgene can be positionedat the 3′ end of the gene of interest, or at the 3′end of a 3′regulatory sequence, e.g., a 3′ untranslated region (UTR) or a 3′flanking sequence. Chromatin opening elements can be flanking on one orboth ends of the expression cassette, e.g., placed 5′ of the expressioncassette.

[0252] Exemplary insulators include a DNA segment which encompasses the5′ end of the chicken β-globin locus and corresponds to the chicken 5′constitutive hypersensitive site as described in PCT Publication94/23046, elements described in Bell et al. (2001) Science 291:447-50.

[0253] Internal Ribosome Entry Sites (IRES). IRES enable eukaryoticribosomes to enter and scan an mRNA at a position other than the 5′ m⁷G-cap structure. If position internally, e.g., 3′ of a first codingregion (or cistron), an IRES will enable translation of a second codingregion within the same transcript. The second coding region isidentified by the first ATG encountered after the IRES. Exemplary IRESelements include viral IRES such as the picornavirus IRES and thecardiovirus IRES (see, e.g., U.S. Pat. No. 4,937,190) and non-viral IRESelements found in 5′ UTRs (e.g. those elements of transcripts encodingimmunoglobulin heavy chain binding protein (BiP) (Macejak, D. G., et al.Nature, 35390-4, 1991); Drosophila Antennapedia (Oh, S. K., et al.,Genes Dev, 6:1643-53, 1992) and Ultrabithorax (Ye, X., et al., Mol. CellBiol., 17:1714-21, 1997); fibroblast growth factor 2 (Vagner, S., etal., Mol. Cell Biol., 15:35-44, 1995); initiation factor eIF4G (Gan, etal., J Biol. Chem., 273:5006-12, 1998); proto-oncogene c-myc (Nanbru, etal., J. Biol. Chem., 272:32061-6, 1995; Stoneley, M., Oncogene,16:423-8, 1998); and vascular endothelial growth factor (VEGF) (Stein,I., et al., Mol. Cell Biol., 18:3112-9, 1998).

[0254] Localization Signals. A “localization signal” is a polypeptidesequence that determines the subcellular localization of polypeptide orprotein. The subcellular localization may be: cytoplasmic, nuclear,nuclear envelope, transmembrane, plasma membrane, plasma membrane outerleaflet, endoplasmic reticulum, Golgi, lysosomal, receptor-coated pits,and so forth. For example, the peptide sequence KDEL (SEQ ID NO:32) isan endoplasmic reticulum localization signal and when grafted onto aheterologous protein results in its endoplasmic reticulum localization.A reformatted protein can be localized within a cell by attachment of alocalization signal, e.g., a localization signal described by Marasco(2001) Curr Top Microbiol Immunol. 260:247-70.

[0255] In combination with appropriate flanking sequences, the antibodyligands can be reformatted such that they are expressed as transmembraneproteins on the surfaces of the cells (e.g., lymphocytes) to program thecells to interact with the target recognized by the antibody ligands. Inthe case of cytotoxic T cells, the ligands may elicit a cytotoxicresponse against the target. The programmed cytotoxic T cells are testedin vitro or in vivo for cytotoxicity.

[0256] Selectable Markers. The recombinant expression vector includesprimary and secondary selectable markers. Primary markers, for example,can be used to select for transformants of mammalian cells. Secondarymarkers, for example, can be used to select for amplification of thetransformed nucleic acid. Exemplary markers include neo, for neomycin orG418 resistance, and HPRT. The choice of selectable markers may dependon the host cell and/or implementation. The DHFR gene, for example, canbe used to select for vector amplification by growing cells (e.g., CHOcells) with increasingly stringent methotrexate selection.

[0257] Leader Sequences. Eukaryotic leader sequences are designed forthe translocation of nascent polypeptides from ribosomes in thecytoplasm directly into the lumen of the endoplasmic reticulum. Leadersequences, typically hydrophobic, include a sequence that is recognizedand cleaved by eukaryotic signal peptidases. The cleavage event producesa mature polypeptide that, absent other signals, is secreted from thecell.

[0258]FIG. 8 illustrates a profile for a eukaryotic, gram negative andgram positive leader sequences. Prokaryotes also detect leader sequenceswhich direct translocation of nascent polypeptides into the periplasm.FIG. 8 also illustrates profiles for the signal sequences of Gramnegative and positive bacteria.

[0259] In some embodiments, the leader sequence includes the sequenceVHS (Valine-Histidine-Serine), or VHA (Valine-Histidine-Alanine) at the−3, −2, and −1 positions respectively, where the position after the −1position is cleaved by signal peptidase. In some instances the sameregion within the leader sequence may be cleaved by both eukaryotic andprokaryotic processing enzymes, such as is the case for a mutatedimmunoglobulin leader ‘MGWSCIILFLVATATGVHA’ (SEQ ID NO:33) sequence,which is functional in mammalian CHO cells, and a mutated M13-pIIIderived leader ‘VKKLLFAIPLVVPFYSVHA’ (SEQ ID NO:34) that is functionalin E. coli. Such areas of amino acid sequence within a leader can beused to position a restriction enzyme site at the 5′ end of a codingsequence to allow expression/secretion-compatible shuttling of codingregions between different expression hosts.

[0260] Isolation of Antibody-Expressing Cells

[0261] Reformatted nucleic acids are introduced into mammalian hostcells, e.g., using conventional transfection techniques, includingcalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, or electroporation. Inaddition, biological vectors, e.g., viral vectors can be used asdescribed below. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001), and other suitablelaboratory manuals. Stable transfected host cells are selected.

[0262] Mammalian cells that produce at least a threshold amount ofantibody can be isolated by FACS (Fluorescence Activated Cell Sorting).The selection compensates, e.g., for variability in expression levelsamong cells, allows isolation of cells with high expression levels. Atleast two types of protocols can be used for antibody production:transient expression protocols and stable expression protocols. FACSselection is typically used to select stable transfected cells.

[0263] Transient expression protocols are useful for initial functionaltesting. Cells, e.g., HekT and COS cells, are transfected with plasmidDNA encoding an individual antibody. The antibody is then purified fromthe medium in which the cells are grown, e.g., without cell sorting.See, e.g., Example 3.

[0264] Stable expression protocols generate stable cell lines which canbe amplified for large-scale antibody production. For example, NSO andCHO cells can be used to generate stable cell lines. In someembodiments, stable cell lines for a number of different cell lines isdone in batch format. The nucleic acid constructs produced by the batchreformatting are introduced into cells, e.g., by transfection orelectroporation. The cells are grown under non-selective conditions, anddiluted—to a limited extent. Then selective conditions are applied toselect for stable cell lines. Cells that grow are screened by FACS (seebelow), magnetic particle-based separation, and/or ELISA to identifyexpressing clones. Such clones, particularly ones that demonstrate highlevel expression, are expanded and used to produce antibody.

[0265] In a related method, stable cell lines are produced bycultivating cells, e.g., CHO cells in suspension, directly in productionmedia (e.g., CHO sera-free medium, e.g. CHO-S-SFM2 from InvitrogenCorp.). These cells are transfected with nucleic acid encoding thedesired antibody. Clones isolated from the transformation are useddirectly for high-density production, e.g., without having to “adapt”the clones to the production medium.

[0266] In one embodiment, the cell lines are also engineered to anenzyme that increases the amount of bisected complex oligosaccharidesthat are added to the Fe region of an antibody. For example, the cellhave increased expression of the β-(1,4)-N-acetylglucosaminyltransferaseIII enzyme can produce antibodies with improved anti-tumor properties(see, e.g., Umana et al. (1999) Nature Biotechnol. 17:176-180).

[0267] The method can also include using CHO cells that have beentransfected with a vector for expressing whole antibodies. The CHO cellsare also modified such that they have the ability to bind antibodies ontheir cell surface, e.g. by a surface expressed IgG binding protein(e.g. a membrane anchored Fe—Receptor or Protein A). Thus, antibodiesproduced by the CHO cells are bound to the surface of the cell.

[0268] To perform FACS sorting, the transfected and modified CHO cellscan be cultured in a low permeability media. The low permeability mediacan be Phosphate Buffered Saline (PBS) containing about 40% gelatin withor without fetal calf serum. The low permeability media reducesdiffusion of the secreted antibodies into the culture, thereby allowingthe secreted antibodies to bind to the surface of the CHO cell fromwhich they are expressed rather than diffuse and bind to another cell.The cells are then removed from the low permeability media and exposedto labeled antibodies that selectively bind a portion of the secretedantibody that is not bound to the surface of the cell. The labeledantibody (which binds the secreted antibody) can be conjugated with afluorophore or a metalisized label. The cells are sorted based ondetection of the labeled antibody, e.g., by using fluorescence activatedcell sorting (FACS) or magnetic cell sorting, respectively. Using FACSor magnetic cell sorting, the level of antibodies secreted and attachedto the CHO cell is detected and those cells which secrete high levels ofhuman antibodies are selected on an individual basis.

[0269] With respect to FACS, the cells are sorted using a fluorescentactivated cell sorter (e.g., a sorter available from Becton DickinsonImmunocytometry Systems, San Jose Calif.; see also U.S. Pat. Nos.5,627,037; 5,030,002; and 5,137,809). As each cell passes through thesorter, a laser beam excites fluorescent compounds that may be attachedto the cell. A detector assesses the amount of light emitted by suchfluorescent compounds, if present. The amount of label bound to eachcell is quantified and, if at least a threshold amount of label isdetected, an electrostatic field is generated to deflect the cell fromits default path. Deflected cells are thus separated and collected. As aresult, cells with low or no antibody expression can be discarded andcells that demonstrate high level antibody expression can be harvestedand cultured.

[0270] A variety of methods can be used to attach secreted antibodies tothe expressing cell prior to the detection phase.

[0271] In one embodiment, antibody secreted by the transfected cells ischemically attached to the surface of the host cell by abiotin-streptavidin linkage. Turning now to FIG. 13, the host cell whichis being assayed for antibody expression is cell-surface biotinylatedwith sulfo-NHS-LC-biotin or sulfosuccinimidyl-6 (biotinamide) hexanoate.The biotin can also be attached e.g., to sialic acid residues by firstadding a ketone, e.g., using N-levulinoyl mannosamine, and thenconjugating the ketone to a biotin hydrazide, e.g.,biotinamido-caproylhydrazide, under physiologic conditions. See, e.g.,Both et al., 2001, Biotechnol. Bioeng 71, 266-73.

[0272] A streptavidin (or avidin) linked moiety such as ProteinA-streptavidin is then bound to the cell surface. The moiety includes anantibody capture agent, e.g., Protein A, Protein G or an antibody thatrecognizes the heavy or light chain of the secreted antibody. Thesecreted antibodies are thus retained on the cell surface. A conjugatedantibody is used detect the retained, secreted antibodies. For example,the conjugate antibody includes a detectable label such as a fluorophorefor FACS analysis or a metalisized label for magnetic particle-basedseparation. A second conjugated antibody used to label human antibodiesattached to the cell surface is selected based upon whether ananti-heavy chain or anti-light chain is used as the ligand-antibody. Ifan anti-heavy chain antibody is used as the capture agent, an anti-lightchain antibody is used as the label conjugated antibody. In embodimentsthat use both the anti-heavy chain and anti-light chain antibodies, onlythose cells that are secreting whole antibodies are identified. Cellswhich only express the light chain or only the heavy chain areundetected.

[0273] In another embodiment, secreted antibodies are attached to thesurface of an expressing cell by a Fc receptor protein. All tester cellsinclude a nucleic acid that directs expression of the Fcγ receptor,i.e., the nucleic acid encoding the Fcγ receptor is operably linked to aregulatory sequence that is active in the tester cells or inducible inthe tester cells. The Fcγ receptor that is expressed on the cell surfacebinds to the heavy chain of the antibodies being expressed. The complexis detected by a third antibody, a conjugated antibody specific for thehuman antibody light chain. For example, the conjugate antibody caninclude a fluorophore for FACS analysis or a metalisized label formagnetic particle-based separation.

[0274] Bioreactors

[0275] The antibody expressing cells can be transferred to a high celldensity culture system, e.g., a system that produces about 100 mg ofantibody using cell densities of about 10⁷ to 10⁸ per ml. In someimplementations, it is useful to produce large quantities (e.g., inexcess of 100 mg) of a few select antibodies. Likewise, in some otherimplementations, it is useful to produce smaller quantities (e.g., from5 mg to 70 mg) of a larger number of antibodies, but still with highyield.

[0276] Membrane Technology. In one embodiment, cells are cultured in acontainer with “cell line membrane technology” (e.g., from Biointegra.)The container includes at least two compartments which are separated bya semi-permeable membrane. Nutrients and other small molecules can crossthe membrane in order to feed cells in the cell compartment. Largemolecules, e.g., those with a molecular weight of greater than 10 kDa(such as antibodies) are retained in the cell compartment. The cells inthe cell compartment rest on a gas exchange surface which exchangesoxygen and carbon dioxide across its surface. These conditions enablehigh cell concentrations in a small volume.

[0277] Hollow-Fiber Reactor. In one embodiment, a hollow fiber reactoris used. Such reactors are available from commercial suppliers, e.g.,Cellex Bioscience. The reactor includes an extracapillary space and anintracapillary space. The cells are grown in the extracapillary spacewhile medium is circulated through the reactor. Secreted antibody isharvested from the medium as it exits the reactor. See FIG. 15 andExample 5, for an example of human antibody production in a hollow fiberbioreactor. In a related aspect, so-called “artificial kidneys” can beused as bioreactors.

[0278] Functional Activity Assays

[0279] The antibodies and other mammalian expressed ligands witheffector domains can be assayed for functional activity either in vitroor in vivo. Information from cell-based assays can be collected asso-called secondary functional information.

[0280] Immunological Assays. Some functional assays can monitor anactivity that depends on an arm of the immune system. Examples includethe following.

[0281] In vitro assays for immunoglobulin effector domain activity,e.g., cytotoxic activity is used to detect the ability of a ligand todeliver antibody effector functions against a target. For example, cellculture assays can be used to assay complement dependent cytotoxicity(CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) mediatedby a reformatted antibody. One ADCC assay is described below.

[0282] The Cr-release assay, for example, can be used to assaycell-mediated cytotoxicity. Peripheral blood lymphocytes (PBL) areprepared as effector cells, while target cells that express the targetedmolecule are loaded with ⁵¹Cr. The target cells are washed and thenseeded into a flat bottom microtitre plate. PBLs are added to the targetcells in combination with the ligand (e.g., a candidate ligand). Maximumrelease is determined by the addition of Tween-20 to target cells,whereas minimal release is determined in the absence of PBLs. Afterovernight incubation, ⁵¹Cr released into the supernatant is counted in aγ scintillation counter.

[0283] In vivo assays include injecting a reformatted antibody into ananimal, e.g., an animal model of a diseased state. For example, theanimal can be a transgenic animal, e.g., expressing an oncogene in aparticular tissue. In another example, the animal is a mouse with axenograft of tumor cells (e.g., human tumor cells). The efficacy of theantibody (or other ligand) can be assayed by comparing time, size, andnumber of tumors formed compared to untreated or control-treatedanimals. In an implementation in which the xenografted mouse is a nudemouse, the mouse can be injected with human PBLs to reconstitute theimmune system. Other physiological parameters of the reformattedantibody can also be monitored including immunogenicity, clearance, andso forth.

[0284] Cellular Activity Assays. Other cellular activity assays includeassessments of cellular pH and calcium flux, and assessments of acellular behavior, e.g., apoptosis, cell migration, cell proliferation,and cell differentiation. Assays can monitor a specific response, e.g.,activation (such as phosphorylation of a transcription factor such asNKκB, and so forth. Other assays are specific for a particular targetcompound. Numerous cell culture assays for differentiation andproliferation are known in the art. Some examples are as follows:

[0285] Assays for embryonic stem cell differentiation (which willidentify, among others, proteins that influence embryonicdifferentiation hematopoiesis) include, e.g., those described in:Johansson et al. (1995) Cellular Biology 15:141-151; Keller et al.(1993) Molecular and Cellular Biology 13:473-486; McClanahan et al.(1993) Blood 81:2903-2915.

[0286] Assays for lymphocyte survival/apoptosis (which will identify,among others, proteins that prevent apoptosis after superantigeninduction and proteins that regulate lymphocyte homeostasis) include,e.g., those described in: Darzynkiewicz et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., CancerResearch 53:1945-1951, 1993; Itoh et al., Cell 66:233 243, 1991;Zacharchuk, Journal of Immunology 145:4037 4045, 1990; Zamai et al.,Cytometry 14:891-897, 1993; Gorczyca et al., International Journal ofOncology 1:639-648, 1992.

[0287] Assays for proteins that influence early steps of T-cellcommitment and development include, without limitation, those describedin: Antica et al., Blood 84:111-117, 1994; Fine et al., CellularImmunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995;Toki et al., Proc. Nat. Acad. Sci. USA 88:7548-7551, 1991.

[0288] Dendritic cell-dependent assays (which will identify, amongothers, proteins expressed by dendritic cells that activate naiveT-cells) include, without limitation, those described in: Guery et al.,J. Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

[0289] Assays for T-cell or thymocyte proliferation include withoutlimitation those described in: Current Protocols in Immunology, Ed by J.E. coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley Interscience (Chapter 3, Tnvitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494 3500,1986; Bertagnolli et al., J. Immunol. 145:1706 1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I.Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,1994.

[0290] Assays for cytokine production and/or proliferation of spleencells, lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. Coligan eds. Vol 1pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurementof mouse and human interleukin gamma., Schreiber, R. D. In CurrentProtocols in Immunology., Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wileyand Sons, Toronto. 1994.

[0291] Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205 1211,1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse andhuman interleukin-6, Nordan, R. In Current Protocols in Immunology. J.E. e.a. Coligan eds. Vol 1 pp. 6.6.1 6.6.5, John Wiley and Sons,Toronto. 1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861,1986; Measurement of human Interleukin-11, Bennett, F., Giannotti, J.,Clark, S.C. and Turner, K. J. In Current Protocols in Immunology.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;

[0292] Assays for T-cell clone responses to antigens (which willidentify, among others, proteins that affect APC-T cell interactions aswell as direct T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Puh. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

[0293] Other assays, for example, can determine biological activity withrespect to endothelial cell behavior, nerve cell growth, nerve cellmigration, spermatogenesis, oogenesis, apoptosis, endocrine signaling,glucose metabolism, amino acid metabolism, cholesterol metabolism,erythropoiesis, thrombopoeisis, and so forth.

[0294] Cell Binding Assays. The functionality of a reformatted antibodycan also be used to in a cell binding assay. The antibody can be labeledbound to a population of cells that includes cells that present a targetrecognized by the antibody. The population can also include cells thatdo not present the target, or that present a related molecule that isdiscriminated by the reformatted antibody.

[0295] In a first example, the reformatted antibody is tested using FACSanalysis. The reformatted antibody is labeled with a fluorophore, eitherdirectly or using a secondary antibody and bound to cells. Then, thecells are passed through a FACS apparatus to count the number of cellsbound by the reformatted antibody. The cells can also be contacted withanother antibody labeled with a fluorophore that is detectable using adifferent channel. Binding of this antibody can be correlated on acell-by-cell basis with binding of the reformatted antibody (e.g., usinga 2D scatter plot).

[0296] In a second example, the reformatted antibody is assayed usingimmunohistochemistry. The antibody is contacted to a histologicalsection. The section is washed, and bound antibody is detected, e.g.,using standard methods.

[0297] In a third example, the reformatted antibody is assayed in vivo,e.g., in a subject organism. The antibody is labeled, e.g., with a NMRcontrast reagent or other traceable reagent. The antibody isadministered to the subject and, after an appropriate interval, itslocalization within the subject is detected, e.g., by imaging thesubject organism.

[0298] Biochemical Assays. Examples of biochemical assays for testingthe functionality of a reformatted antibody include: Western blotanalysis, immunoprecipitations, and other binding assays such as surfaceplasmon resonance (SPR).

[0299] SPR or Biomolecular Interaction Analysis (BIA) detectsbiospecific interactions in real time, without labeling any of theinteractants. Changes in the mass at the binding surface (indicative ofa binding event) of the BIA chip result in alterations of the refractiveindex of light near the surface (the optical phenomenon of surfaceplasmon resonance (SPR)). The changes in the refractivity generate adetectable signal, which are measured as an indication of real-timereactions between biological molecules. Methods for using SPR aredescribed, for example, in U.S. Pat. No. 5,641,640; Raether (1988)Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal.Chem. 63:2338-2345; Szabo et al (1995) Curr. Opin. Struct. Biol.5:699-705.

[0300] Information from SPR can be used to provide a quantitativemeasure of the equilibrium dissociation constant (K_(d)), and kineticparameters, including K_(on) and K_(off), for the binding of a ligand toa target. Such data can be used to compare different ligands. Forexample, reformatted antibodies selected from the library can becompared to identify individuals that have high affinity for the targetor that have a slow K_(off). This information can also be used todevelop structure-activity relationships (SAR).

[0301] Affinity Maturation/Optimization

[0302] At any stage after initial identification, a selected librarymember can be mutagenized to improve its binding affinity or any otherproperty. For example, a first display library is used to identify oneor more ligands for a target. These identified ligands are then mutatedto form a second display library. Higher affinity ligands are thenselected from the second library, e.g., by using higher stringency ormore competitive binding and washing conditions.

[0303] Numerous techniques can be used to mutate the identified ligands.These techniques include: error-prone PCR (Leung et al. (1989) Technique1:11-15), recombination, DNA shuffling using random cleavage (Stemmer(1994) Nature 389-391), RACHITT™ (Coco et al (2001) Nature Biotech.19:354), site-directed mutagenesis (Zoller et al. (1987) MethodsEnzymol. 1987;154:329-50.; Zoller et al. (1982) Nucl Acids Res10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) MethodsEnzymol. 208:564-586) and incorporation of degenerate oligonucleotides(Griffiths et al (1994) EMBO J 13:3245).

[0304] For antibodies, mutagenesis can be directed to the CDR regions ofthe heavy or light chains. Further, mutagenesis can be directed toframework regions near or adjacent to the CDRs. Mutagenesis canintroduce synthetic or natural diversity.

[0305] Another exemplary method for introducing diversity in a mannerguided by the original sequence is the hybridization-directed methoddescribed in provisional application No. 60/343,954, filed Oct. 24,2001, “HYBRIDIZATION CONTROL OF SEQUENCE VARIATION”.

[0306] Targets

[0307] Targets. Generally, any molecular species can be used as atarget. In some embodiment, more than one species is used as a target,e.g., a sample is exposed to a plurality of targets. The target can beof a small molecule (e.g., a small organic or inorganic molecule), apolypeptide, a nucleic acid, cells, and so forth.

[0308] One class of targets includes polypeptides. Examples of suchtargets include small peptides (e.g., about 3 to 30 amino acids inlength), single polypeptide chains, and multimeric polypeptides (e.g.,protein complexes).

[0309] A polypeptide target can be modified, e.g., glycosylated,phosphorylated, ubiquitinated, methylated, cleaved, disulfide bonded andso forth. Preferably, the polypeptide has a specific conformation, e.g.,a native state or a non-native state. In one embodiment, the polypeptidehas more than one specific conformation. For example, prions can adoptmore than one conformation. Either the native or the diseasedconformation can be a desirable target, e.g., to isolate agents thatstabilize the native conformation or that identify or target thediseased conformation.

[0310] In some cases, however, the polypeptide is unstructured, e.g.,adopts a random coil conformation or lacks a single stable conformation.Agents that bind to an unstructured polypeptide can be used to identifythe polypeptide when it is denatured, e.g., in a denaturing SDS-PAGEgel, or to separate unstructured isoforms of the polypeptide forcorrectly folded isoforms, e.g., in a preparative purification process.

[0311] Some exemplary polypeptide targets include: cell surface proteins(e.g., glycosylated surface proteins or hypoglycosylated variants),cancer-associated proteins, cytokines, chemokines, peptide hormones,neurotransmitters, cell surface receptors (e.g., cell surface receptorkinases, seven transmembrane receptors, virus receptors andco-receptors, extracellular matrix binding proteins such as integrins,cell-binding proteins (e.g., cell attachment molecules or “CAMs” such ascadherins, selectins, N-CAM, E-CAM, U-CAM, I-CAM and so forth), or acell surface protein (e.g., of a mammalian cancer cell or a pathogen).In some embodiments, the polypeptide is associated with a disease, e.g.,cancer.

[0312] The target polypeptide is preferably soluble. For example,soluble domains or fragments of a protein can be used. This option isparticularly useful for identifying molecules that bind to transmembraneproteins such as cell surface receptors and retroviral surface proteins.

[0313] Some exemplary targets include: cell surface proteins (e.g.,glycosylated surface proteins or hypoglycosylated variants),cancer-associated proteins, cytokines, chemokines, peptide hormones,neurotransmitters, cell surface receptors (e.g., cell surface receptorkinases, seven transmembrane receptors, virus receptors andco-receptors, extracellular matrix binding proteins, cell-bindingproteins, antigens of pathogens (e.g., bacterial antigens, malarialantigens, and so forth).

[0314] More specific examples include: integrins, cell attachmentmolecules or “CAMs” such as cadherins, selections, N-CAM, E-CAM, U-CAM,I-CAM and so forth); proteases, e.g., subtilisin, trypsin, chymotrypsin;a plasminogen activator, such as urokinase or human tissue-typeplasminogen activator (t-PA); bombesin; factor IX, thrombin; CD-4;CD-19; CD20; platelet-derived growth factor; insulin-like growthfactor-I and -II; nerve growth factor; fibroblast growth factor (e.g.,aFGF and bFGF); epidermal growth factor (EGF); transforming growthfactor (TGF, e.g., TGF-α and TGF-β); insulin-like growth factor bindingproteins; erythropoietin; thrombopoietin; mucins; human serum albumin;growth hormone (e.g., human growth hormone); proinsulin, insulin A-chaininsulin B-chain; parathyroid hormone; thyroid stimulating hormone;thyroxine; follicle stimulating hormone; calcitonin; atrial natriureticpeptides A, B or C; leutinizing hormone; glucagon; factor VIII;hemopoictic growth factor; tumor necrosis factor (e.g., TNF-α andTNF-β); enkephalinase; mullerian-inhibiting substance;gonadotropin-associated peptide; tissue factor protein; inhibin;activin; vascular endothelial growth factor; receptors for hormones orgrowth factors; protein A or D; rheumatoid factors; osteoinductivefactors; an interferon, e.g., interferon-α,β,γ; colony stimulatingfactors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs),e.g., IL-1, IL-2, IL-3, IL-4, etc.; decay accelerating factor;immunoglobulin (constant or variable domains); and fragments of any ofthe above-listed polypeptides. In some embodiments, the target isassociated with a disease, e.g., cancer.

[0315] Cells as Targets. Another class of targets includes cells, e.g.,fixed or living cells. The cell can be bound to an antibody that iscovalently attached to a paramagnetic particle or indirectly attached(e.g., via another antibody). For example, a biotinylated rabbitanti-mouse Ig antibody is bound to streptavidin paramagnetic beads and amouse antibody specific for a cell surface protein of interest is boundto the rabbit antibody.

[0316] In one embodiment, the cell is a recombinant cell, e.g., a celltransformed with a heterologous nucleic acid that expresses aheterologous gene or that disrupts or alters expression of an endogenousgene. In another embodiment, the cell is a primary culture cell isolatedfrom a subject, e.g., a patient, e.g., a cancer patient. In stillanother embodiment, the cell is a transformed cell, e.g., a mammaliancell with a cell proliferative disorder, e.g., a neoplastic disorder. Instill another embodiment, the cell is the cell of a pathogen, e.g., amicroorganism such as a pathogenic bacterium, pathogenic fungus, or apathogenic protist (e.g., a Plasmodium cell) or a cell derived from amulticellular pathogen.

[0317] Cells can be treated, e.g., at a particular stage of the washingstep. The treatment can be a drug or an inducer of a heterologouspromoter-subject gene construct. The treatment can cause a change incell behavior, morphology, and so forth. Molecules that dissociate fromthe cells upon treatment are collected and analyzed.

[0318] Examples of cells include, a cancer cell, a hematopoietic cell,BalI cells, primary culture cells, malignant cells, neuronal cells,embryonic cells, placental cells, and non-mammalian cells (e.g.,bacterial cells, fungal cells, plant cells) and so forth. Cancer cells,for example, are attached to magnetically responsive particles using anantibody specific for a marker on the cell surface, e.g., CD19 or acell-surface cancer-specific antigen.

[0319] In a preferred embodiment, the cells are recombinant cells. Thecells can be transformed with a plasmid that expresses (e.g., undercontrol of an inducible or constitutive promoter) a cell-surface proteinof interest. The plasmid can also express a marker protein, e.g., foruse in binding the transformed cell to a magnetically responsiveparticle. In another embodiment, the cells express an intracellularprotein, e.g., an oncogene, transcription factor, or cell-signalingprotein. The intracellular protein can alter cell behavior or therepertoire of molecules on the cell surface. In still anotherembodiment, the cells are treated (e.g., using a drug or geneticalteration) to alter the rate of endocytosis, pinocytosis, exocytosis,and/or cell secretion.

[0320] Still more exemplary targets include organic molecules. In oneembodiment, the organic molecules are transition state analogues and canbe used to select for catalysts that stabilize a transition statestructure similar to the structure of the analogue. In anotherembodiment, the organic molecules are suicide substrates that covalentlyattach to catalysts as a result of the catalyzed reaction.

[0321] Effector Domains

[0322] Effector domains can be attached to the antibody ligand duringthe reformatting process. One exemplary effector domain is a polypeptidethat includes an immunoglobulin constant region, e.g., an Fc domain.

[0323] Fc domains. As discussed above, Fc domains mediate effectorfunctions by recruiting Clq for complement-dependent cytotoxicity (CDC)and FcγRs for ADCC.

[0324] The Fc region of IgG molecules is glycosylated at asparagine 297in the CH2 domain. This asparagine is the site for modification withbiantennary-type oligosaccharides. It has been demonstrated that thisglycosylation is required for effector functions mediated by Fcγreceptors and complement Clq (Burton and Woof (1992) Adv. Immunol.51:1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76). In a preferredembodiment, the Fc domain is produced in a mammalian expression systemthat appropriately glycosylates the residue corresponding to asparagine297 (Kabat numbering). The Fc domain can also include other eukaryoticmodifications.

[0325] The Fc domain can be attached to the hinge region, which is foundbetween CH1 and CH2 of antibody heavy chains. The hinge region canimpart a flexible structure that facilitates the recruitment of effectorfunctions which bind in the CH2 domain in the proximity of the hingeregion and also, e.g., antigen aggregation by a second antigen bindingdomain.

[0326] In one embodiment, the Fc domain is a modified Fc domain. Forexample, the Fc domain can be altered, e.g., such that it has alteredbinding properties (e.g., enhanced or diminished). For example, the Fcdomain can be engineered to preferentially binding to some Fc receptorsrelative to others. Shields et al. (2001) J Biol Chem 276:6591-6604describes a variant IgG1 Fc domain that has improved binding toFcγRIIIA. Idusogie et al. (2000) J. Immunol. 164:4178 describes an IgG1mutant that alters Clq binding and complement activation.

[0327] In still another embodiment, the effector domain is a syntheticpolypeptide that binds to an Fc receptor or to complement. Suchsynthetic polypeptides can be identified by a phage display selectionfor 6 to 20 amino acid cyclic peptides that specifically binding to onespecies of Fc receptor, but not another.

[0328] Non-Immunological Effector domains. In some embodiments,non-immunological effector domains, including effector domains composedof polypeptides and polypeptide conjugates. Examples of such effectordomains include the following labels and cytotoxins.

[0329] Labels. For example, the effector fragment can include apolypeptide label or a non-polypeptide label. Polypeptide labels includeenzymes, such as horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase. Other polypeptide labelsinclude luciferase, luciferin, aequorin, and green fluorescent protein(and its derivatives). For example, an effector domain fragment thatincludes GFP can be used to identify the localization of a target in asample, e.g., a histological sample.

[0330] Cytotoxins. Polypeptide and non-polypeptide cytotoxins can beused as an effector domain. Examples of polypeptide cytotoxins includediphtheria toxin, cholera toxin, abrin, pseudomonas exotoxin, and ricinA.

[0331] Display Libraries

[0332] A display library is a collection of entities; each entityincludes an accessible polypeptide component and a recoverable componentthat encodes or identifies the polypeptide component. The polypeptidecomponent can be of any length, e.g. from three amino acids to over 300amino acids. In some embodiments, the ligand discovery platformdescribed herein uses a display library as a diverse source of potentialligands.

[0333] A variety of formats can be used for display. The following aresome examples.

[0334] Phage Display. One format utilizes viruses, particularlybacteriophages. This format is termed “phage display.” The polypeptidecomponent is typically covalently linked to a bacteriophage coatprotein. The linkage results form translation of a nucleic acid encodingthe polypeptide component fused to the coat protein. The linkage caninclude a flexible peptide linker, a protease site, or an amino acidincorporated as a result of suppression of a stop codon. Phage displayis described, for example, in Ladner et al., U.S. Pat. No. 5,223,409;Smith (1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO90/02809; de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboomet al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) ImmunolToday 2:371-8; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991)Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods Enzymol.267:129-49; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982.

[0335] Phage display systems have been developed for filamentous phage(phage f1, fd, and M13) as well as other bacteriophage (e.g. T7bacteriophage and lambdoid phages; see, e.g., Santini (1998) J. Mol.Biol. 282:125-135; Rosenberg et al. (1996) Innovations 6:1-6; Houshmandet al. (1999) Anal Biochem 268:363-370). The filamentous phage displaysystems typically use fusions to a minor coat protein, such as gene IIIprotein, and gene VIII protein, a major coat protein, but fusions toother coat proteins such as gene VI protein, gene VII protein, gene IXprotein, or domains thereof can also been used (see, e.g., WO 00/71694).In a preferred embodiment, the fusion is to a domain of the gene IIIprotein, e.g., the anchor domain or “stump.”

[0336] The valency of the polypeptide component can also be controlled.Cloning of the sequence encoding the polypeptide component into thecomplete phage genome results in multivariant display since allreplicates of the gene III protein are fused to the polypeptidecomponent. For reduced valency, a phagemid system can be utilized. Inthis system, the nucleic acid encoding the polypeptide component fusedto gene III is provided on a plasmid, typically of length less than 700nucleotides. The plasmid includes a phage origin of replication so thatthe plasmid is incorporated into bacteriophage particles when bacterialcells bearing the plasmid are infected with helper phage, e.g. M13K01.The helper phage provides an intact copy of gene III and other phagegenes required for phage replication and assembly. The helper phage hasa defective origin such that is the helper phage genome is notefficiently incorporated into phage particles relative to the plasmidthat has a wild type origin.

[0337] Bacteriophage displaying the polypeptide component can be grownand harvested using standard phage preparatory methods, e.g. PEGprecipitation from growth media.

[0338] After selection of individual display phages, the nucleic acidencoding the selected polypeptide components, by infecting cells usingthe selected phages. Individual colonies or plaques can be picked, thenucleic acid isolated and sequenced.

[0339] Peptide-Nucleic Acid Fusions. Another format utilizespeptide-nucleic acid fusions. Polypeptide-nucleic acid fusions can begenerated by the in vitro translation of mRNA that include a covalentlyattached puromycin group, e.g., as described in Roberts and Szostak(1997) Proc. Natl. Acad. Sci. USA 94:12297-12302, and U.S. Pat. No.6,207,446. The mRNA can then be reverse transcribed into DNA andcrosslinked to the polypeptide.

[0340] Cell-based Display. In still another format the library is acell-display library. Proteins are displayed on the surface of a cell,e.g., a eukaryotic or prokaryotic cell. Exemplary prokaryotic cellsinclude E. coli cells, B. subtilis cells, spores (see, e.g., Lu et al.(1995) Biotechnology 13:366). Exemplary eukaryotic cells include yeast(e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hanseula, orPichia pastoris). Yeast surface display is described, e.g., in Boder andWittrup (1997) Nat. Biotechnol. 15:553-557 and U.S. Provisional PatentApplication No. Serial No. 60/326,320, filed Oct. 1, 2001, titled“MULTI-CHAIN EUKARYOTIC DISPLAY VECTORS AND THE USES THEREOF.” Thisapplication describes a yeast display system that can be used to displayimmunoglobulin proteins such as Fab fragments, and the use of mating togenerate combinations of heavy and light chains.

[0341] In one embodiment, variegate nucleic acid sequences are clonedinto a vector for yeast display. The cloning joins the variegatedsequence with a domain (or complete) yeast cell surface protein, e.g.,Aga2, Aga1, Flo1, or Gas1. A domain of these proteins can anchor thepolypeptide encoded by the variegated nucleic acid sequence by atransmembrane domain (e.g., Flo1) or by covalent linkage to thephospholipid bilayer (e.g., Gasl). The vector can be configured toexpress two polypeptide chains on the cell surface such that one of thechains is linked to the yeast cell surface protein. For example, the twochains can be immunoglobulin chains.

[0342] Ribosome Display. RNA and the polypeptide encoded by the RNA canbe physically associated by stabilizing ribosomes that are translatingthe RNA and have the nascent polypeptide still attached. Typically, highdivalent Mg²⁺ concentrations and low temperature are used. See, e.g.,Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes etal. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) MethodsEnzymol. 328:404-30. and Schaffitzel et al. (1999) J Immunol Methods.231(1-2):119-35.

[0343] Other Display Formats. Yet another display format is anon-biological display in which the polypeptide component is attached toa non-nucleic acid tag that identifies the polypeptide. For example, thetag can be a chemical tag attached to a bead that displays thepolypeptide or a radiofrequency tag (see, e.g., U.S. Pat. No.5,874,214).

[0344] Scaffolds. One typical scaffold for ligand discovery is anantibody (e.g., Fab fragments, single chain Fv molecules (scFV), singledomain antibodies, camelid antibodies, and camelized antibodies).However, aspects of the ligand discovery platform described herein canbe applied to the discovery of ligands that depend on other types ofscaffolds for a structural framework.

[0345] Another example of a small scaffolding domain is a so-called“cysteine loop” formed by a pair of cysteines separated by amino acids,e.g., between three and 25 amino acids, or between four and ten aminoacids. The intervening amino acids can be any amino acid other thancysteine, in which case, under oxidizing conditions, the pair ofcysteines disulfide bond and constrain the topology of the interveningamino acids. Randomized short linear peptides, e.g., between four and 25amino acids, or between four and 15 amino acids, can also be used.

[0346] Still other exemplary scaffolds can include: T-cell receptors;MHC proteins; extracellular domains (e.g., fibronectin Type III repeats,EGF repeats); protease inhibitors (e.g., Kunitz domains, ecotin, BPTI,and so forth); TPR repeats; trifoil structures; zinc finger domains;DNA-binding proteins; particularly monomeric DNA binding proteins; RNAbinding proteins; enzymes, e.g., proteases (particularly inactivatedproteases), RNase; chaperones, e.g., thioredoxin, and heat shockproteins.

[0347] Appropriate criteria for evaluating a scaffolding domain caninclude: (1) amino acid sequence, (2) sequences of several homologousdomains, (3) 3-dimensional structure, and/or (4) stability data over arange of pH, temperature, salinity, organic solvent, oxidantconcentration. In one embodiment, the scaffolding domain is a small,stable protein domains, e.g., a protein of less than 100, 70, 50, 40 or30 amino acids. The domain may include one or more disulfide bonds ormay chelate a metal, e.g., zinc.

[0348] Examples of small scaffolding domains include: Kunitz domains (58amino acids, 3 disulfide bonds), Cucurbida maxima trypsin inhibitordomains (31 amino acids, 3 disulfide bonds), domains related to guanylin(14 amino acids, 2 disulfide bonds), domains related to heat-stableenterotoxin IA from gram negative bacteria (18 amino acids, 3 disulfidebonds), EGF domains (50 amino acids, 3 disulfide bonds), kringle domains(60 amino acids, 3 disulfide bonds), fungal carbohydrate-binding domains(35 amino acids, 2 disulfide bonds), endothelin domains (18 amino acids,2 disulfide bonds), Streptococcal G IgG-binding domain (35 amino acids,no disulfide bonds) and small intracellular signaling domains such asSH2, SH3, and EVH domains. Generally, any modular domain, intracellularor extracellular, can be used.

[0349] Further display technology can also be used to obtain ligandsthat are particular epitopes of a target. This can be done, for example,by using competing non-target molecules that lack the particular epitopeor are mutated within the epitope, e.g., with alanine. Such non-targetmolecules can be used in a negative selection procedure as describedbelow, as competing molecules when binding a display library to thetarget, or as a pre-elution agent, e.g., to capture in a wash solutiondissociating display library members that are not specific to thetarget.

[0350] Automated Methods and Information Management

[0351] Any and all aspects of the ligand identification platform can beautomated. Referring now to FIG. 14, information, particularlyfunctional information and tracked events associated with liganddiscovery is stored in a central database 260. For example, the database260 can include primary functional information 230 (such as data onbinding properties from ELISAs) as well as secondary functionalinformation 250 (such as data on ADCC activity of a mammaliancell-expressed ligand). Instances of each datum are associated withinstances of library members.

[0352] The database server 260 can also track events associated with:the initial selection of library members for binding to a target; samplehandling of library nucleic acids that are being advanced forreformatting 240; expression levels of library members in mammaliancells; and functional activity of proteins (e.g., 250) isolated frommammalian expression systems.

[0353] The automation and information management strategies enable therapid screening of numerous individual library members and then winnowthese to a more limited number of ligands. In some cases, the use ofautomation to perform the selection increases the reproducibility of theselection process as well as the through-put. Further, the cohesive andhighly refined monitoring enables operators to finely tune the discoveryprocess.

[0354] After selecting a pool of target binding members from a displaylibrary, the identified members of the pool are individually isolatedusing a robotic device. Referring now to FIG. 15 and FIG. 16, for aphage display library, for example, the pool can be infected intobacterial cells which are then plated 316 at a density such thatindividual colonies or plaques are formed from each infection event. Theindividual colonies are picked 320 into wells of a multi-well plate,e.g., a 94- or 364-well plate, using an automated colony picker.Typically, the colonies are picked in duplicate, e.g., intocorresponding wells of two identical plates. One of the plates isarchived. The other can be used as a source for subsequent analyses.

[0355] Automated picking enables the picking of at least 100, 10³, 10⁴,10⁵ (or more) selected library members. Each of these library memberscan then by analyzed individually as described below. Information aboutthe picking can be stored in the database, e.g., using cross-referencedrecords for, respectively, the plate, each well, and each displaylibrary member.

[0356] Assays. Referring again to FIG. 15 and FIG. 16, the individuallibrary members are analyzed using an assay 324, typically a highthrough-put assay. The assay determines functional information for thepolypeptide component being displayed for each library member. Thefunctional information can be obtained for the polypeptide componentwhen it is either attached or removed from the library vehicle, e.g.,the bacteriophage. The functional information is recorded in thedatabase 60 in a table of assay results. Each entry in the tableincludes a field that points to the display library member being assayedand another field that stores the result of the assay, and otherrelevant information such as background levels, and results forcontrols. A variety of possible assays, including binding assays (suchas ELISAs) can be used to obtain functional information about binding.

[0357] Equipment. Various robotic devices can be employed in theautomation process. These include multi-well plate conveyance systems,magnetic bead particle processors, liquid handling units, and colonypicking units.

[0358] These devices can be built on custom specifications or purchasedfrom commercial sources, such as Autogen (Framingham Mass.), BeckmanCoulter (USA), Biorobotics (Woburn Mass.), Genetix (New Milton,Hampshire UK), Hamilton (Reno Nev.), Hudson (Springfield N.J.),Labsystems (Helsinki, Finland), Packard Bioscience (Meriden Conn.), andTecan (Mannedorf, Switzerland).

[0359] The database server 260 can also be configured to communicatewith each device using commands and other signals that are interpretableby the device. The computer-based aspects of the system can beimplemented in digital electronic circuitry, or in computer hardware,firmware, software, or in combinations thereof. An apparatus of theinvention, e.g., the database server 260, can be implemented in acomputer program product tangibly embodied in a machine-readable storagedevice for execution by a programmable processor; and method actions canbe performed by a programmable processor executing a program ofinstructions to perform functions of the invention by operating on inputdata and generating output. One non-limiting example of an executionenvironment includes computers running Windows NT 4.0 (Microsoft) orbetter or Solaris 2.6 or better (Sun Microsystems) operating systems.

[0360] Viral Vectors for Expression

[0361] In one embodiment, nucleic acids encoding the ligands arereformatted by incorporation into a viral vector for viral-base deliveryinto cells in culture or in a subject organism. Viral vector systemsinclude those of DNA and RNA viruses, e.g., retroviruses, lentiviruses,adenoviruses, and herpes simplex viruses. Adeno-associated virus, lentivirus, and retroviral systems enable integration of the nucleic acidencoding the ligands into a chromosome.

[0362] Viruses the incorporate the reformatted nucleic acids encodingligands can be used to generated target-directed cytotoxic T cells. Forexample, viruses with tropism for lymphoid cells are prepared as vectorsand used to infect cytotoxic T lymphocytes. For example, plasma membraneassociated antibody ligands can be expressed in the T lymphocytes.

[0363] Reformatting for Yeast Display Libraries

[0364] In another embodiment, a yeast display library that encodesheteromultimeric ligands, such as antibodies (e.g., Fabs) is produced byreformatting a phage display library. Again, the process can beperformed en masse. Referring to FIG. 19, the ApaL1 to NotI fragment ofthe phage display vector is inserted into a yeast display libraryvector. The fragment contains sequences encoding VL and CL, for thelight chain, a sequence encoding a leader sequence, VH, CH, and a myctag, for the heavy chain. A prokaryotic ribosomal binding site islocated between the AscI and SfiI sites as is the sequence encoding theleader for the heavy chain. Insertion of the ApaL1-NotI fragment into ayeast display vector positions the yeast GAL1 promoter and a sequenceencoding a yeast leader sequence (i.e., signal sequence) and an anchorprotein (such as Aga2p or a fragment thereof) 5′ of the coding strand ofthe fragment and a myc tag and stop codon C-terminal.

[0365] The segment intervening between the light and heavy chain codingregions is then substituted by removing the prokaryotic ribosomalbinding site and leader sequence and inserting a sequence that includesa stop codon, the yeast GAL1 promoter, a yeast leader sequence, and asequence encoding an anchor protein (such as Aga2p or fragment thereof).The yeast leader sequence can also be the signal peptide of Aga2p. Aga2pis a subunit of the a-agglutinin protein). The reformatting locates theanchor protein at the N-terminus of both subunits and results in twotranscriptional units operably linked to yeast promoters.

[0366] Yeast display library members can also be reformatted forexpression in a phage display vector, e.g., by reversing the stepsdescribed above. See also U.S. Provisional Patent Application Serial No.60/326,320, filed Oct. 1, 2001, titled “MULTI-CHAIN EUKARYOTIC DISPLAYVECTORS AND THE USES THEREOF.”

[0367] Further, yeast display library members can be reformatted forexpression in mammalian cells. The ApaL1-Not fragment from the yeastvector is transferred into pBRV. Then, an AscI-SfiI or AscI-MfeIfragment is substituted to insert an IRES or a mammalian promoter, suchas the CMV promoter. The reformatting also removes the N-terminal Aga2panchor protein so that antibodies produced in mammalian cells have anative N-terminus or a short peptide addition at the N-terminus (e.g., 6residues or less).

[0368] Reformatting scFV Coding Sequences

[0369] A library of nucleic acids encoding scFv antibodies can bereformatted as sequences encoding Fabs or even complete IgG antibodiesusing the methods described herein. Unique restriction enzyme sites arepositioned 5′ and 3′ of the sequence encoding the linker between the VLand VH domains in the scFv on the coding strand. First the nucleic acidfragment that includes the sequences encoding the VL and VH domain andthe intervening linker are transferred to a Fab expression vector ormammalian expression vector. For the example above, a Fab expressionvector includes a sequence encoding the CH1 domain so that the VHencoding sequence is inserted immediately 5′, with respect to the codingstrand, to form a sequence encoding VH-CH1. Then the unique restrictionsites flanking the intervening sequence are used to replace the sequenceencoding the peptide linker with a fragment that includes a sequenceencoding a CL domain, a stop codon, and any necessary interveningregulatory sequences such as a prokaryotic ribosome binding site, anIRES, a eukaryotic promoter, and/or an eukaryotic leader sequence.

[0370] This reformatting process can also be reversed. The expression ofscFv antibodies can be useful, for example, in expression systemssusceptible to non-stoichiometric production of heavy and light chainsor poor association of the heavy and light chains.

[0371] Still other reformatting applications may be relevant. Forexample, antibody encoding sequences can be reformatted for expressionin plants, e.g., using plant virus (such as the Cauliflower MosaicVirus) promoters, and a plant signal sequences. See, e.g., U.S. Pat. No.6,080,560.

[0372] The following invention is further illustrated by the followingexamples, which should not be construed as further limiting. Thecontents of all references, pending patent applications and publishedpatents, cited throughout this application are hereby expresslyincorporated by reference. This provisional application also includes anAppendix which is an integral component of the application, andaccordingly the Appendix incorporated in its entirety.

EXAMPLE 1 Restriction-Based Shuttling of Fab Cassettes Between Bacterialand Mammalian Expression Vectors

[0373] Bacterial and mammalian expression vectors were prepared thatsupport the transfer individually or en masse of Fab heavy and lightchain genes from a bacterial expression vector to a mammalian expressionvector while maintaining linkage between paired heavy and light chaingenes.

[0374] Referring to FIG. 2 (top), the Fab expression cassette of adisplay vector that serves as a source of Fab genes for reformatting(top), can include the following elements: a bacterial promoter (P),followed by a bacterial leader sequence (L) which in turn is followed bycloning sites, including ApaL1, AscI, MfeI, BstEII, and NotI. Sequencesencoding an immunoglobulin light chain (VL and CL) are inserted betweenthe ApaL1 and AscI sites. These sequences are followed by a stop codon(not shown), a bacterial ribosomal binding site (rbs), and a secondleader sequence (L). The MfeI site is positioned in the FRI region of anantibody variable region encoding sequence and downstream of the leadersequence. Sequences encoding an immunoglobulin heavy chain fragment (VHand CHI) are inserted between the MfeI and NotI sites. Alternatively,sequences encoding an immunoglobulin heavy chain fragment (VH) areinserted between the MfeI and BstEII sites. These sequences are in framewith a sequence encoding a filamentous bacteriophage gene III coatprotein (III). The display vector also contains selectable markers andorigins of replication (not shown).

[0375] Typically, the display vector is a phagemid or phage displayvector, which mediate the expression of the Fab on the surface of thebacteriophage M13 or fd. The Fab-encoding segment is transferred fromthe bacterial display vector to the eukaryotic vector, e.g., pBRV orpRRV (FIG. 3) by restricting the vector with ApaL1 and BstE2. Thisfragment is subcloned into ApaL1/BstE2 sites of pBRV or pRRV.Alternatively, if the Fab cassette also carries a unique NheI sitewithin the CH1 domain, the cloning can be done via this site instead ofBstEII. This vector contains a CMV eukaryotic promoter in place of thebacterial promoter, and a eukaryotic leader sequence in place of thefirst bacterial leader sequence. The VH-CH1 sequence is no longer fusedto gene III but is fused in-frame to a sequence encoding animmunoglobulin Fe region, e.g., including Hinge-CH2-CH3. Thisconstruction encodes a full length IgG heavy chain. The vector alsoincludes a selectable neomycin resistance gene and sequences formaintenance in bacteria (see FIG. 3).

[0376] The bacterial RBS is replaced with a eukaryotic IRES byrestricting the vector with AscI and MfeI and replacing the excisedfragment (which includes the bacterial RBS) with a fragment thatincludes the IRES and a second eukaryotic leader sequence is inserted.This vector is transformed into a eukaryotic host and the encodedcomplete antibody is assayed for functional activity.

[0377] pBRV and pRRV are related vectors that differ in the position andreading frame of the ApaL1 at the 3′ end of the leader driving lightchain secretion. Antibody genes transferred to pBRV in tworestriction-cloning steps encode antibodies that include an additionalalanine residue at the N-terminus of VL after processing. Antibody genestransferred to pRRV in a procedure in which the Fab fragment is excisedfrom their source display construct by PCR, have an N-terminus that isunaltered relative a naturally-occurring VL.

EXAMPLE 2 Leader Sequences

[0378] Referring to FIG. 4, two possible intervening segments which canbe inserted between heavy and light chain coding sequences are depicted.Both segments include an IRES between the EcoRI and XbaI site forinternal ribosome entry and translation of the second coding region. Oneintervening segment, from pblue (top), includes the R27080 leader,MARRLWILSLLAVTLTVALAEVQL (SEQ ID NO: 1). Cleavage of this leadersequence follows the final alanine and releases a mature N-terminus thatbegins with EVQL (SEQ ID NO:3). The other, from pRRV, includes anantibody leader sequence, MGWSCIILFLVATATGAHSEVQL (SEQ ID NO:2). Thisleader sequence is cleaved after the final serine and releases a matureN-terminus that begins with EVQL (SEQ ID NO:3).

EXAMPLE 3 PCR-Based Shuttling of Fab Cassettes Between Bacterial andMammalian Expression Vectors

[0379] The Fab genes in bacterial expression constructs are amplifiedwith designed PCR primers that include suitable restriction enzymerecognition sites at positions compatible for transfer, e.g., at theborder of V genes. The amplified PCR products are digested with ApaL1and NheI and cloned into the pRRV vector, digested with ApaL1 and Nhe. Asecond step is used to replace the region between the heavy and lightchain genes with a eukaryotic IRES and eukaryotic leader sequence.Expression of the antibodies from the pRRV vector results in productionof full-length antibodies that include an N-terminus that is unalteredrelative to a naturally-occurring VL.

EXAMPLE 4 Transient Expression

[0380] For a given target, twenty Fabs are identified from a displaylibrary. These Fabs are reformatted into IgG form. Each IgG is expressedand purified to obtain about 200 μg to 500 μg of antibody. The processinvolves: batch transfer, re-identification of initial Fabs, transientexpression of individual clones, and purification.

[0381] Each IgG is expressed by, transient expression in HekT cellsgrown in ten 10 cm diameter culture dishes. For each IgG, about 100 mlto 200 ml conditioned culture media are obtained. Each IgG is purifiedfrom the harvested media.

[0382] The method described can be up-scaled for production of up to 10mgs of antibody. In addition, other methods, based on transienttransfection of cells grown at high cell density in bioreactors usingviral or non-viral vectors (Curr Opin Biotechnol 1999 April;10(2):156-9), can be used. Expression levels of >20 mg/liter have beendescribed for Hek293 cells, grown in 1-3 liter bioreactors andtransfected with an expression construct of a human IgG1 byCa-phosphate-DNA co-precipitate technology (Biotechnol Bioeng(2001)75:197-203).

EXAMPLE 5 Isolation of High Expressing Cells from a Pool of StableTransfectants using

[0383] Stable clones of antibody expressing cells are isolated asfollows. Cells transfected with antibody expression constructs are grownin a medium of low permeability. The antibodies that they express arecaptured at the surface of the secreting cell. These captured antibodiesare detected on the surface associated soluble antibody with afluorescent dye conjugated secondary reagent. The sample (i.e., the cellpool) is analyzed in a flow cytometer and sorted to isolate thesub-population (1-5% of cells) with highest expression levels.

[0384] This subpopulation can be subjected to one or more cycles ofsorting and/or cultivated to establish clonal cell lines that producehigh levels of antibody expression.

[0385] Applications of the method are as follows:

[0386] 1. isolation of high expressing cells from a pool of cellstransfected with an individual IgG construct;

[0387] 2. isolation of high expressing cells for many different IgGantibodies from a pool of cells transfected with a mixture of constructs(i.e., constructs for the different IgG antibodies); and

[0388] 3. isolation of high(er) expressing cells obtained bymethotrexate amplification of IgG constructs in CHO dhfr-cells (e.g., asdescribed in Borth et al. (2001) Biotechnol. Bioeng. 71:266-73

EXAMPLE 6

[0389] The term “CJ library” refers to an exemplary naive library.

[0390] This example describes tools and strategies for reformatting ofFabs to whole human IgG antibodies. The reformatting vectors arecompatible with a number of antibody display libraries; the strategiesallow, e.g., fast reformatting of Fabs to IgGs.

[0391] These reformatting strategies can be used for the fast assemblyof a mammalian expression vector (FIG. 6 depicts pRRV), for simultaneousexpression of both chains of a human IgG antibody. Light chain (LC) andheavy chain (HC) expression are under control of the same promoter, thetwo “open reading frames” are linked via an “internal ribosome entrysite” (IRES). Besides the antibody expression cassette, the constructcontains the neomycin resistance gene as selectable marker forgeneration of stable cell lines. The SV40 origin of replication allowsamplification of the vector in SV40 transformed (SV40 large T antigenexpressing) cells, thereby facilitating increased antibody expressionlevels in transient expression systems, in e.g. HekT cells.

[0392] pRRV, the preferentially used expression vector for reformattedCJ Fabs is shown in FIG. 6.

[0393] Two exemplary approaches include: a) reformatting of Fabfragments from the display vector/phage into the eukaryotic expressionvector pBRV can be done by restriction endonuclease/enzyme cloning; andb) reformatting of Fab fragments from the display vector/phage into theeukaryotic expression vector pRRV by a PCR based strategy.

[0394] Reformatting into pBRV (Batch Reformatting Vector):

[0395] Due to compatibility of restriction sites in the displayconstructs and pBRV, the transfer of Fabs can be performed solely byrestriction fragment cloning (“cut and paste”). Besides reformatting ofan individual Fab, it is also possible to batch transfer (see below) Fabfragments derived from a mixture of simultaneously propagated (e.g.,without PCR) phages/phagemids. After a “cut and paste” transfer of Fabsinto pBRV, it is not necessary to verify the complete DNA sequence ofthe “whole antibody” constructs obtained, since no PCR step has beeninvolved.

[0396] Reformatting into pRRV (Rapid Reformatting Vector):

[0397] For reformatting of individual Fabs to “unaltered” antibodies aPCR based strategy can be used. Unmodified human IgG antibodies with“natural” N-termini are obtained. A verification of the complete DNAsequence is required, after reformatting. Reformatting of Fabs from a“non-CJ” library requires the design of two V gene specific primers foreach individual clone. In case of the CJ library a very small number ofPCR primers is sufficient for amplification of virtually all Fabspresent in the library. It is also possible to batch transfer Fabs fromthe CJ library to unaltered human antibodies.

[0398] Batch reformatting is the simultaneous transfer of a Fabrepertoire into an IgG expression plasmid. The linkage betweenpreviously selected LC/HC pairs needs to be retained. This is achievedin a first cloning step, in which the complete Fab fragment istransferred into the eukaryotic expression construct. In a secondcloning step, regulatory elements, important for HC expression, areintroduced (see sections 6.1, 6.2 and 6.4). The second step is notrelated to retaining of the linkage between pre-selected LC/HC pairs.When a mixture of Fabs is batch reformatted, it is necessary to analyzea significantly higher number of output antibody clones, compared toinput Fabs, to ensure their re-identification.

[0399] It is typically possible to generate antibodies with natural HCsequences from Fabs derived from the CJ library, no matter ifreformatted into pBRV (6.2) or pRRV (6.1). This is due to the presenceof a unique Asc1 site, introduced at the 5′ end of the VH region of eachCJ Fab. Asc1 rarely cuts within antibody V genes. Due to this uniquefeature of the CJ library, the transfer of an individual Fab to pRRV (asdescribed in 6.1) can be performed in a “batch reformatting type”procedure; in a first step the complete Fab, lifted from the phage byPCR, is inserted into pRRV.

[0400] Preferred procedure for batch reformatting of CJ Fabs tounaltered IgG antibodies:

[0401] Two special features of the CJ library, already mentioned above,facilitate batch reformat a mixture of Fabs to unaltered antibodies, byuse of PCR:

[0402] 1. Possibility to obtain natural human antibody HC by “cut andpaste” (without DNA sequence modification).

[0403] 2. Only a very small number of VL specific primers (1 kappa, 4lambda; see 6.1) are needed for amplification of all Fabs contained inthe library. At the same time, these primers also introduce “natural” LCN-termini.

[0404] In addition, PCR amplification of Fab fragments from the phagesmight be more feasible than propagation of al large number of phages,and isolation of a sufficient amount of phage DNA to perform “cut andpaste” reformatting. Individual Fabs are amplified from phage inseparate, parallel PCR reactions. The combined mixture of Fabfragments/PCR products will be batch transferred into pRRV, using theapproach described in 6.1 for an individual CJ Fab. To re-identify the“starting Fabs”, a significantly higher number of antibody constructshave to be sequenced. The Fab portion of the reformatted antibody can besequenced to verify PCR fidelity.

[0405] 6.1 Transfer of Individual CJ Fabs to pRRV

[0406] In a first step the CJ Fab is lifted from the display vector viaPCR. One primer, containing an Nhe1 site, anneals to the CH1 region ofthe HC. This Nhe1 site is compatible to the one introduced into the CH1region in the antibody expression vectors, pRRV and pBRV. The otherprimer contains an ApaL1 site, (compatible with correct processing ofthe eukaryotic leader/signal peptide (L) of pRRV) and anneals to the 5′end of VL. Because of conservation of N-terminal amino acid sequences ofλ and κ LC of CJ Fabs, only a small number (5) of VL specific primersare needed to amplify most Fab clones of the CJ library. The ApaL1/Nhe1cut PCR fragment of about 1. 1 kb is inserted into pRRV.

[0407] In a second step internal/regulatory elements are exchanged viaAsc1 and Mfe1. The Asc1/Asc1 fragment encoding IRES and an “Ab-leader”sequence (fragment size: ˜0.7 kb) should be taken from pShuttleI. (Aninternal fragment carrying the Ab-leader and a more efficient IRES2element, as judged from recent/preliminary expression experiments, canbe taken from pShuttleIII.) As explained above, it is also possible totransfer a mixture of CJ Fabs in batch to pRRV, after separate PCRamplification of individual clones.

[0408] Recommended PCR Primers for Amplification of CJ Fabs: Primersspecific for VL genes of the CJ library: κ:         ApaL1 5′-ATATAT GTGCAC TCT GAC ATC CAG ATG ACC CAG TC (SEQ ID NO:35)                     V   H   S   D   I   Q   M   T   Q   S (SEQ IDNO:36) λ:         ApaL1 5′-ATATAT GTG CAC TCA CAG AGC GTC TTG ACT C (SEQID NO:37)            V   H   S   Q   S   V   L   T (SEQ ID NO:38)           ApaL1 5′-ATATAT GTG CAC TCA CAG AGC GCT TTG ACT C (SEQ IDNO:39)            V   H   S   Q   S   A   L   T (SEQ ID NO:40)           ApaL1 5′-ATATAT GTG CAC TCA AGC TAC GAA TTG ACT C (SEQ IDNO:41)            V   H   S   S   Y   E   L   T (SEQ ID NO:42)           ApaL1 5′-ATATAT GTG CAC TCA CAG AGC GAA TTG ACT C (SEQ IDNO:43)            V   H   S   Q   S   E   L   T (SEQ ID NO:44)

[0409] The sequence underlined has to be added to the N-terminus of thelight chain. The sequence “ATATAT” represents a variable overhang,facilitating efficient ApaL1 cleavage of the PCR product. CH1 specificprimer:              NheI 5′-GGA GGG TGC TAG CGG GAA GAC CG-3′ (SEQ IDNO:45)

[0410] A mismatch at the position of the introduced Nhe1 site does notinfluence the performance of this primer.

[0411] Recommended PCR conditions: (e.g., using 2Advantage HF polymerasefrom Clontech.) 25-30 cycles of:

[0412] 1 min. annealing at 55° C.

[0413] 1.5 min. extension at 68° C.

[0414] 30 sec. denaturation at 96° C.

[0415] 6.2 Transfer of CJ Fabs to PBRV by “Cut and Paste”

[0416] Referring to FIG. 5, in the first step an ApaL1/BstE2 fragment ofabout 1.1 kb is released from the display construct and inserted intopBRV. (The ApaL1 site in pBRV corresponds to the one in the displayvector. This compatibility leads to the addition of one alanine-residueto the “natural” N-terminus of the LC.). In a second stepinternal/regulatory elements are exchanged via Asc1 and Mfe 1. TheAsc1/Mfe1 fragment, encoding IRES and an “Ab-leader” sequence (fragmentsize: ˜0.7 kb), should be taken from pShuttleI. (An internal fragmentcarrying the Ab-leader and a IRES2 element can be taken frompShuttleIII.). The method can be adapted, e.g., for individual Fabs andFab-repertoires.

[0417] 6.3 Transfer of Individual TQ Fabs to pRRV

[0418] Referring to FIGS. 18 and 19, in a first step the LC and the VHregion of the Fab are amplified from the display vector by PCR. Uponcutting with appropriate restriction endonucleases, the LC is insertedinto pRRV and VH into the Shuttle vector, respectively. In case of theLC one primer has to be specific for the 5′ end of the VL region of theindividual clone and will also introduce the ApaL1 site, compatible withcorrect processing of the eukaryotic leader/signal peptide (L) of pRRV.The second primer has to be “display vector specific”. The LC isinserted into pRRV as ApaL1/Asc1 fragment (˜0.6 kb).

[0419] For VH amplification one primer has to be specific for the 5′ endof VH, and will also introduce a BssH2 site, compatible with correctprocessing of the eukaryotic leader/signal peptide (L) included in theShuttle vector. The second primer anneals to the CH1 region of the Fab.This primer also contains an Nhe1 site, compatible to the one introducedinto the CH region, present in the antibody expression vectors, pRRV andpBRV. VH is inserted in pShuttleII as BssH2/Nhe1 fragment (˜0.4 kb).

[0420] In the second step, the Mlu1/Nhe1 fragment of pShuttleII (+VH) isinserted into pRRV (+LC). The ˜1.0 kb fragment excised from the Shuttlevector also includes the IRES motif. The pRRV construct has to be cutwith Asc1 and Nhe1. Asc1 and M1u1 create compatible 5′ overhangs.

[0421] In the procedure outlined above the pShuttleIII vector can beused in place of pShuttleII. This constructs contains the IRES2 motifthat can contribute to higher antibody expression levels.

[0422] PCR Amplification of Vgenes of an Individual TQ-Fab:

[0423] This method can use, e.g., “clone specific” forward/senseprimers. Furthermore, the VL and VH specific primers need to includeadditional sequences, compatible to the leader sequences in pRRV andpShuttle, respectively. The junctions of leader sequences and VgeneN-termini are shown below: pRRV: ApaLI GGCGTGCACTCT (SEQ ID NO:46) G  V  H  S-VL (SEQ ID NO:47) pShuttle: BssHJL GGCGCGCACTCC (SEQ IDNO:48)  G  A  H  S-VH (SEQ ID NO:49) The reverse antisense primers bindto the plasmid backbone of the display vector (pCES): For LCamplification one can use a primer binding in the “ribo- some bindingsite”: 5′-TCC AGC GGC TGC CGT AGG CAA TAG-3′ (pCESrbrev) (SEQ ID NO:50)For VH amplification the CH1 specific primer mentioned in 6.1 can beused:             NheI 5′-GGA GGG TGC TAG CGG GAA GAC CG-3′ (SEQ IDNO:51)

[0424] 6.4 Transfer of TQ Fabs to pBRV by “Cut and Paste”

[0425] (Individual Fabs and Fab-Repertoires)

[0426] Referring to FIG. 20, in the first step an ApaL1/BstE2 fragment(˜1.1 kb) is released from the display construct and inserted into pBRV.(The ApaL1 site in pBRV is corresponding to the one in the displayvector. This compatibility leads to the addition of one alanine-residueto the “natural” N-terminus of the LC.) In a second step internalregulatory elements are exchanged via Asc1 and Sfi1. The Asc1/Sfi1fragment (˜0.7 kb) can be taken from pBlue/IRES/Sfi1. This fragmentcontains the IRES motif and a modified “viral signal peptide” (derivedfrom HCMV). Because of utilization of the Sfi1 site of the prokaryoticPelB leader, the six C-terminal amino acids “AQPAMA” (SEQ ID NO:52) ofthe PelB leader are included in the eukaryotic signal peptide.

[0427] 6.5 Variations of Reformatting of Individual Fabs (CJ or TQ) intopBRV

[0428] This example can be used, e.g., to transfer Fabs with additionalBstE2 sites.

[0429] As described in 6.2 and 6.4 Fabs from the present and the newDyax library are cloned into pBRV using ApaL1 and the BstE2 (unique inVH, cuts in FR4). Unfortunately, BstE2 also cuts in a portion (˜10-20%)of VL genes. Two alternative strategies for the transfer of such Fabsinto pBRV are shown below:

[0430] Variant 1: “Cut and paste” transfer in 3-steps—VH is insertedinto the vector before LC. Referring to FIG. 21, the construct isassembled in an order that does not conflict with additional BstE2 sitesin VL: 1) An Asc1/BstE2 fragment (˜0.4-0.5 kb) of the Fab insert of thephage or phagemid is cloned into pBRV. This fragment contains VH. 2)insertion of the LC into pBRV as ApaL1/Asc1 fragment (˜0.6 kb). 3)Introduction of eukaryotic regulatory elements (Asc1/Asc1 fragment forCJ Fabs and Asc1/Sfi1 fragment for TQ Fabs) (fragment size: ˜0.7 kb)

[0431] Variant 2: Cloning of a PCR-amplified Fab into pBRV. Referring toFIG. 5, the Fab (˜1.1 kb) is lifted from the phage using vector/backbonespecific PCR primer. One primer binds 5′ of VL, the other in CH1. TheCH1 specific primer also contains an Nhe1 site, compatible to the oneintroduced in the CH1 region within the antibody expression vector pBRV.The Fab is inserted into pBRV as ApaL1/Nhe1 fragment. The exchange ofregulatory sequences (second step) is performed as described in 6.1 and6.2. (A ˜0.7 kb fragment encoding the IRES motif and an Ab-leader isintroduced.)

[0432] PCR primers:

[0433] Forward primer binding in LacZ promoter: (example)

[0434] 5′-AGC GGA TAA CAA TTT CAC ACA GG-3′ (SEQ ID NO:53)

[0435] Reverse primer binding in CH1 (including a Nhe1 site,underscored): (see also 6.1)

[0436] 5′-GGA GGG TGC TAG CGG GAA GAC CG-3′ (SEQ ID NO:54)

[0437] Recommended PCR conditions:

[0438] 25-30 cycles of:

[0439] 1 min. annealing at 55° C.

[0440] 1.5 min. extension at 68° C.

[0441] 30 sec. denaturation at 96° C.

[0442] 7.1 Recommended Sequencing Primers for “Clone Verification” andSequencing of Complete Fab Fragments, Reformatted into pBRV or pRRV:

[0443] Primers that bind in the CMV promoter and the CH1 region and thatare useful for clone verification include, for example:

[0444] pCMV forward primer:

[0445] 5′-CGC AAA TGG GCG GTA GGC GTG-3′ (SEQ ID NO:55)

[0446] (reading into 5′-VL)

[0447] CH1 (reverse/antisense):

[0448] 5′-GTC CTT GAC CAG GCA GCC CAG GGC-3′ (SEQ ID NO:56)

[0449] (reading into 3′-VH)

[0450] These primers anneal within the internal fragment, e.g. the IRESmotif, which is introduced in the second reformatting step.

[0451] Alternatively, sequencing of the entire Fab is possible after thefirst reformatting step. Primers binding to prokaryotic internalsequences (ribosome binding site) can be used:

[0452] pCESrbrev:

[0453] 5′-TCC AGC GGC TGC CGT AGG CAA TAG-3′ (SEQ ID NO:57)

[0454] (reading into the constant region (3′) of the LC)

[0455] pCESrb (forward/sense):

[0456] 5′-GCG CCA ATT CTA TTT CAA GG-3′ (SEQ ID NO:58)

[0457] (reading into 5′-VH)

[0458] Other embodiments are within the following claims.

What is claimed:
 1. A method comprising: (i) providing a plurality ofinitial nucleic acid cassettes comprising a) a first coding regionencoding a first immunoglobulin variable domain, b) a second codingregion encoding a second immunoglobulin variable domain, and c) aribosomal binding site disposed between the first and second codingregions for translation of the second polypeptide in a first expressionsystem, wherein the first and second coding regions are in the sametranslational orientation; (ii) modifying each nucleic acid cassette ofthe plurality in a single reaction mixture so that it is functional in asecond expression system, wherein the first and second region remainphysically attached during the modifying; (iii) introducing eachmodified nucleic acid cassette into a mammalian cell to produce amixture of transfected cells; and (iv) expressing each modified nucleicacid cassette in the transfected cells.
 2. The method of claim 1 furthercomprising: screening the mixture of transfected cells usingfluorescence-activated cell sorting to identify transfected cell thatproduces a least a threshold amount of an immunoglobulin that includesthe combination of first and second immunoglobulin variable domainpresent in an initial cassette.
 3. The method of claim 1 wherein themodifying comprises inserting a segment that comprises an internalribosome entry site between the first and second coding regions.
 4. Themethod of claim 1 wherein the segment further comprises a signalsequence functional in a mammalian cell.
 5. The method of claim 4wherein the segment further comprises a polyA addition regulatorysequence.
 6. The method of claim 1 wherein the modifying comprisesattaching the second coding region in frame to a sequence encoding aconstant domain to form a fusion protein.
 7. The method of claim 6wherein modifying comprises a) replacing a nucleic acid segment betweenthe first and second regions, b) replacing a nucleic acid 5′ of thecoding strand of the first coding region, and c) replacing a nucleicacid 3′ of the coding strand of the second coding region.
 8. The methodof claim 1 wherein the modifying comprises restricting using one or moreof: ApaLI, AscI, MfeI, BstEII, NotI, XbaI, NcoI, PstI, NheI, SfiI andBssH2.
 9. The method of claim 8 wherein the modifying comprisesreleasing a segment between the first and second coding regions usingAscI and MfeI; AscI and SfiI; ApaL1 and NotI; ApaL1 and NheI; or ApaL1and BstEII.
 10. The method of claim 6 wherein the constant domaincomprises an Fc domain.
 11. The method of claim 1 further comprisingscreening the transfected cells by attaching a probe to the surface ofthe cells, the probe having a binding domain that recognizes a constantregion of the fusion proteins, and detecting the interaction between thefusion protein and the surface bound probe.
 12. The method of claim 6wherein the constant domain comprises a cytotoxic domain.
 13. The methodof claim 12 further comprising detecting cytotoxic activity.
 14. Themethod of claim 1 further comprising culturing the cells in a lowpermeability medium.
 15. A method of screening nucleic acids, the methodcomprising: providing a first plurality of different nucleic acids, eachencoding a hetero-oligomeric candidate ligand; selecting a subset of thefirst plurality by contacting candidate ligands encoded by members ofthe first plurality to a target; reformatting each nucleic acid of thesubset for mammalian cell expression, such that each nucleic acidencodes a hetero-oligomeric protein that includes a first functionaldomain of one subunit of the candidate ligand, a second functionaldomain of another subunit of the candidate ligand and an effector domainnot encoded by the nucleic acids of the first plurality; introducingmembers of the subset into a mammalian cell to form a plurality ofexpression cells that can produce the protein that includes thefunctional domain and the effector domain; and screening the expressioncells to identify cells that produce at least a threshold amount of aligand-effector domain fusion protein.
 16. The method of claim 15wherein the screening comprises fluorescence-activated cell sorting. 17.A method for evaluating display library members, the method comprising:providing a plurality of display library members; determining anassessment for each library member with respect to a property; storinginformation about the assessments of the library members in a computerdatabase; filtering the information to identify a subset of the librarymembers; and reformatting each member of the subset for expression in amammalian cell by a method that comprises disposing nucleic acid foreach member of the selected subset into a single container.
 18. Themethod of claim 17 further comprising introducing reformatted membersinto mammalian cells and screening the mammalian cells for production ofa polypeptide encoded by a respective reformatted member.
 19. The methodof claim 17 wherein the display library members comprise phage displaylibrary members.
 20. The method of claim 17 wherein determining anassessment comprises a binding assay.